WO2015036378A1 - Épinards résistants au mildiou - Google Patents

Épinards résistants au mildiou Download PDF

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Publication number
WO2015036378A1
WO2015036378A1 PCT/EP2014/069130 EP2014069130W WO2015036378A1 WO 2015036378 A1 WO2015036378 A1 WO 2015036378A1 EP 2014069130 W EP2014069130 W EP 2014069130W WO 2015036378 A1 WO2015036378 A1 WO 2015036378A1
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Prior art keywords
plant
spinach
resistance
gene
progeny
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PCT/EP2014/069130
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English (en)
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Jan Ane DIJKSTRA
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Nunhems B.V.
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Publication of WO2015036378A1 publication Critical patent/WO2015036378A1/fr

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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
    • A01H5/12—Leaves
    • 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
    • A01H6/02—Amaranthaceae or Chenopodiaceae, e.g. beet or spinach
    • A01H6/028—Spinacia oleracea [spinach]

Definitions

  • the invention relates to a new dominant resistance gene, designated RPF13, for use in breeding downy mildew resistant spinach plants.
  • the present invention further relates to cultivated spinach (Spinacia oleraced) seeds, plants and plant parts (e.g. leaves) grown from the seeds, that are resistant to Peronospora farinosa f.sp. spinaciae (abbreviated herein as Pfs) due to the presence of RPF13 in their genome, as well as to progenies of the plants and propagation material for producing the plants.
  • Pfs Peronospora farinosa f.sp. spinaciae
  • Pfs race 1 (Pfs:01 or Pfsl) was first reported in 1824 and resistance to race 1 was identified later in two Egyptian accessions (PI 140467 and PI 140464) and incorporated into commercial hybrid varieties, such as Califlay (Smith and Zahara, California Agriculture, July 1956). In 1958 race 2 appeared and a few years later a single dominant gene imparting resistance against both race 1 and race 2 was identified (Smith et al. 1961 and 1962). In 1976 race 3 appeared and again several years later resistance against race 3 was found. Race 4 was only identified in 1990, and Brandenberger et al. (1992) identified accessions CGN09546, of which 60% of individual plants were resistant, and SPl 82/87, of which 80%) of individual plants were resistant.
  • WO2013/064436 describes a dominant resistance gene, called R6, which confers resistance against Pfsl -6, 9, 11-14 (see Table 1 on page 19 of WO2013/064436; in 2011 the type strain UA4410 has been designated Pfs 14 by the International Working Group on Peronospora farinosa, IWGP).
  • Pfs 14 the International Working Group on Peronospora farinosa
  • no single resistance gene is known, which confers resistance against all known Pfs races, or especially against Pfs races 7-14. It is, therefore, an object of the invention to provide a single dominant resistance gene, which confers resistance against at least Pfs races 7-14.
  • the gene optionally also confers resistance against one or more or all of Pfs races 1-6.
  • the gene also confers resistance against isolate UA4712.
  • the resistance gene designated RPF13, confers resistance against at least Pfs races 7-14, and optionally also against isolate UA4712 and/or optionally against one or more or all of Pfs 1-6.
  • seeds, plants and plant parts or cultivated spinach comprising the new major resistance gene are provided.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • 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.
  • “Spinach” or “cultivated spinach” or “cultivated Spinacia oleracea” refers herein to plants of the species Spinacia oleracea (or seeds from which the plants can be grown), and parts of such plants, bred by humans for food and having good agronomic characteristics.
  • This includes any type of spinach, such as savoy, flat- or smooth-leaf spinach or semi-savoy types. Wild spinach (i.e. not cultivated spinach) or wild relatives of spinach, such as Spinacia tetrandra and Spinazia turkestanica, are not encompassed by this definition.
  • the term "plant” includes the seed (from which the plant can be grown), the whole plant or any parts such as plant organs (e.g., harvested or non-harvested leaves, etc.), plant cells, plant protoplasts, plant cell- or tissue cultures from which whole plants can be regenerated, propagating or non-propagating plant cells, plants cells which are not in tissue culture (but which are for example in vivo in a plant or plant part), plant callus, plant cell clumps, plant transplants, seedlings, plant cells that are intact in plants, plant clones or micro-propagations, or parts of plants (e.g., harvested tissues or organs), such as plant cuttings, vegetative propagations, embryos, pollen, ovules, flowers, leaves, heads, seeds (produced on the plant after self-fertilization or cross-fertilization), clonally propagated plants, roots, stems, stalks, root tips, grafts, parts of any of these and the like, or derivatives thereof,
  • any developmental stage is included, such as seedlings, cuttings prior or after rooting, mature and/or immature plants or mature and/or immature leaves.
  • seeds of a plant these either refer to seeds from which the plant can be grown or to seeds produced on the plant, after self-fertilization or cross-fertilization.
  • somatic cells and “reproductive cells” can be distinguished, whereby somatic cells are cells other than gametes (e.g. ovules and pollen), germ cells and gametocytes. Gametes, germ cells and gametocytes are “reproductive cells”.
  • Tissue Culture or “cell culture” refers to an in vitro composition comprising isolated cells of the same or a different type or a collection of such cells organized into plant tissue. Tissue cultures and cell cultures of spinach, and regeneration of spinach plants therefrom, is well known and widely published (see, e.g. Nguyen et al., 2013, Plant Biotechnology Reports, Vol. 7 Issue 1, p99).
  • Hardested plant material refers herein to plant parts (e.g., leaves detached from the whole plant) which have been collected for further storage and/or further use.
  • Hardvested seeds refers to seeds harvested from a line or variety, e.g., produced after self-fertilization or cross-fertilization and collected.
  • Hardvested leaves refers to spinach leaves, i.e., the plant without the root system, for example substantially all (harvested) leaves.
  • Progeny or “progenies” or “descendants” as used herein refers to offspring, or the first and all further descendants derived from (obtainable from) (derivable from or obtained from) a plant of the invention that comprises (retains) the RPF13 resistance gene in homozygous or heterozygous form and/or the
  • Progeny may be derived by regeneration of cell culture or tissue culture, or parts of a plant, or selling of a plant, or by producing seeds of a plant.
  • progeny may also encompass spinach plants derived from crossing of at least one spinach plant with another spinach plant of the same or another variety or (breeding) line, and/or backcrossing, and/or inserting of a locus into a plant and/or mutation.
  • a progeny is, e.g., a first generation progeny, i.e. the progeny is directly derived from, obtained from, obtainable from or derivable from the parent plant by, e.g., traditional breeding methods (selling and/or crossing) or regeneration.
  • progeny generally encompasses further generations such as second, third, fourth, fifth, sixth, seventh or more generations, i.e., generations of plants which are derived from, obtained from, obtainable from or derivable from the former generation by, e.g., traditional breeding methods, regeneration or genetic transformation techniques.
  • a second generation progeny can be produced from a first generation progeny by any of the methods mentioned above.
  • double haploid plants are progeny.
  • Plant line is for example a breeding line which can be used to develop one or more varieties.
  • 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.
  • Hybrid refers to the seeds harvested from crossing one plant line or variety with another plant line or variety, and the plants or plant parts grown from said seeds.
  • Fl hybrid plant (or Fl hybrid seed) is the generation obtained from crossing two non-isogenic inbred parent lines.
  • Fl hybrid seeds are seeds from which Fl hybrid plants grow.
  • an “interspecific hybrid” refers to a hybrid produced from crossing a plant of one species, e.g. S.
  • oleracia with a plant of another species, e.g. S. tetrandra or S. turkestanica.
  • Crossing refers to the mating of two parent plants. Equally “Cross-pollination” refers to fertilization by the union of two gametes from different plants. “Selfing” refers to the self-pollination of a plant, i.e. to the union of gametes from the same plant.
  • Backcrossing refers to a breeding method by which a (single) trait, such as Pfs resistance conferred by the RPF13 resistance gene, can be transferred from one genetic background (also referred to as "donor”; generally but not necessarily this is an inferior genetic background) into another genetic background (also referred to as "recurrent parent”; generally but not necessarily this is a superior genetic background).
  • An offspring of a cross e.g. an Fl plant obtained by crossing a wild spinach or wild relative of spinach with a cultivated spinach; or an F2 plant or F3 plant, etc., obtained from selfing the Fl
  • the superior genetic background e.g. to the cultivated parent.
  • the trait of the donor genetic background e.g. the RPF13 gene
  • conversion plant or “single locus conversion” in this context refer to plants which are developed by backcrossing wherein essentially all of the desired morphological and/or physiological characteristics of the recurrent parent are recovered in addition to the one or more genes (e.g. the RPF13 resistance gene) transferred from the donor parent.
  • traditional breeding techniques encompasses herein crossing, backcrossing, selling, selection, chromosome doubling, double haploid production, embryo rescue, the use of bridge species, 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, the RPF13- resistance gene can be obtained, identified, selected, and/or transferred.
  • Regeneration refers to the development of a plant from in vitro cell culture or tissue culture or vegetative propagation.
  • Plantative propagation means the method of taking part of a plant and allowing that plant part to form at least roots where plant part is, e.g., defined as or derived from (e.g. by cutting off) leaf, pollen, embryo, cotyledon, hypocotyl, cells, protoplasts, meristematic cell, root, root tip, pistil, anther, flower, shoot tip, shoot, stem, fruit, and petiole.
  • plant part is, e.g., defined as or derived from (e.g. by cutting off) leaf, pollen, embryo, cotyledon, hypocotyl, cells, protoplasts, meristematic cell, root, root tip, pistil, anther, flower, shoot tip, shoot, stem, fruit, and petiole.
  • vegetative propagation it is also referred to as a “vegetative propagation” or a “vegetatively propagated plant”.
  • Single locus converted (conversion) plant refers to plants which are developed by plant breeding techniques comprising or consisting of backcrossing, wherein essentially all of the desired
  • morphological and/or physiological characteristics of a spinach plant are recovered in addition to the characteristics of the single locus (e.g. the locus comprising the RPF13 gene) having been transferred into the plant via the backcrossing technique and/or by genetic transformation.
  • Transgene or “chimeric gene” refers to a genetic locus comprising a DNA sequence which has been introduced into the genome of a spinach plant by transformation.
  • a plant comprising a transgene stably integrated into its genome is referred to as "transgenic plant”.
  • Pfs or “Peronospora farinosd' or “downy mildew” refers to races of the oomycete Peronospora farinosa f.sp. spinaciae.
  • Pfs 1 -Pfs 14 refer to the officially recognized races, which can be differentiated on the differential hosts of spinach and which can be obtained from the Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands, or via references provided by the ISF (International Seed Federation).
  • “Differential hosts” or “differentials” refers to the differential hosts of spinach for distinguishing Pfs races 1-14, which can be obtained from the Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands, or via references provided by the ISF (International Seed Federation).
  • "UA4712” refers to a pathogenic isolate of Pfs which has not yet been officially recognized as a new Pfs race. It is described by Correl and Koike, 2013, Race diversity and the biology of the spinach downy mildew pathogen, CLGRB Annual Report, April 1, 2012 to March 31, 2013.
  • a "Pfs resistant plant” or “downy mildew resistant plant” or a plant having "Pfs resistance” or a “Pfs resistant phenotype” refers to a spinach plant which is resistant against one or more pathogenic races (and pathogenic isolates) of Pfs, as determined in a qualitative resistance assay under controlled environmental conditions.
  • a resistance assay a plurality of plants (e.g. at least 2 replicates of at least 10 plants) of a genotype, are inoculated with a sporangial suspension of the race or isolate and incubated under suitable conditions. After a suitable incubation period (e.g. 7, 8, 9, 10, 11 or more days after inoculation) the plants are evaluated for symptoms.
  • Susceptible controls should show sporulation at the time of symptom evaluation. Any plant showing sporulation on the cotyledons (and/or on the true leaf/leaves) is considered “susceptible", while any plant not showing any sporulation on the cotyledons (and/or on the true leaf/leaves) is considered “resistant”. A plant genotype with more than 85% of the inoculated plants (preferably more than 95%) being classified as "resistant” plant is considered to a resistant against the race or isolate. In the test >85%> of inoculated plants (preferably more than 95% of plants) of the susceptible control plant, such as cultivar Viroflay, should show sporulation. Suitable tests are described herein in the Examples, or in Irish et al.
  • RP 73 refers herein to a single, dominant Pfs resistance gene, which confers Pfs resistance (as defined above) against at least races 7-14, and optionally against UA4712 (and optionally against new pathogenic isolates).
  • RPF13 refers to a resistance gene which confers resistance against at least races 7-14 and optionally further against one or more or all of races 1-6, and/or optionally against UA4712 (and optionally against new pathogenic isolates). The resistance is conferred when the gene is in homozygous or heterozygous form.
  • the resistance phenotype is also referred to herein as the "Pfs resistance phenotype conferred by the RPF13 gene".
  • locus means a specific place or places or a site on a chromosome where for example a gene (e.g. the RPF13 gene) or genetic marker is found.
  • the resistance locus comprising the RPF13 gene is introgressed from a wild spinach or wild relative of spinach.
  • allele(s) means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus.
  • alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome.
  • loci plural locus on a chromosome.
  • One allele is present on each chromosome of the pair of homologous chromosomes.
  • a diploid plant species may comprise a large number of different alleles at a particular locus. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous).
  • gene means a (genomic) DNA sequence comprising a region (transcribed region), which is transcribed into a messenger RNA molecule (mRNA) in a cell, and an operably linked regulatory region (e.g. a promoter).
  • mRNA messenger RNA molecule
  • an operably linked regulatory region e.g. a promoter
  • Different alleles of a gene are thus different alternative forms of the gene, which may be in the form of e.g. differences in one or more nucleotides of the genomic DNA sequence (e.g. in the promoter sequence, the exon sequences, intron sequences, etc.), mRNA and/or amino acid sequence of the encoded protein.
  • Allelism test refers to a genetic test whereby it can be tested whether a phenotype, such as Pfs resistance, seen in two plants, is determined by the same gene or by different genes. For example, the plants to be tested are crossed with each other, the Fl is selfed and the segregation of the phenotypes amongst the F2 progeny is determined. Other segregating populations can equally be made (e.g.
  • the ratio of segregation of the phenotype indicates if the genes are allelic (alleles of the same gene) or non-allelic (different, independent genes).
  • “Introgression fragment” or “introgression segment” or “introgression region” refers to a chromosome fragment (or chromosome part or region) which has been introduced into another plant of the same or related species by crossing or traditional breeding techniques, such as backcrossing, i.e. the introgressed fragment is the result of breeding methods referred to by the verb "to introgress” (such as backcrossing).
  • wild spinach or wild relatives of spinach are often used to introgress fragments of the wild genome into the genome of cultivated spinach.
  • Such a spinach plant thus has a "genome of Spinacia oleracea", but comprises in the genome a fragment of a wild spinach or spinach relative.
  • the introgression fragment never includes a whole chromosome, but only a part of a chromosome.
  • 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 pairs) or less, or about 0.5 Mb (equals 500,000 base pairs) or less, such as about 200,000 bp (equals 200 kilo base pairs) or less, about 100,000 bp (100 kb) or less, about 50,000 bp (50 kb) or less, about 25,000 bp (25 kb) or less.
  • “Physical distance” between loci (e.g. between molecular markers and/or between phenotypic markers) on the same chromosome is the actual physical distance expressed in base pairs (bp), kilo base pairs (kb) or megabase pairs (Mb).
  • Genetic distance between loci is measured by frequency of crossing-over, or recombination frequency (RF) and is indicated in centimorgans (cM).
  • RF recombination frequency
  • cM centimorgans
  • One cM corresponds to a recombination frequency of 1%. If no recombinants can be found, the RF is zero and the loci are either extremely close together physically or they are identical. The further apart two loci are, the higher the RF.
  • a genetic element, a locus, an introgression fragment or a gene or allele conferring a trait (such as resistance against Pfs) is said to be “obtainable from” or can be “obtained from” or “derivable from” or can be “derived from” or “as present in” or “as found in” a plant or seed if it can be transferred from the plant or seed in which it is present into another plant or seed in which it is not present (such as a line or variety) using traditional breeding techniques without resulting in a phenotypic change of the recipient plant apart from the addition of the trait conferred by the genetic element, locus, introgression fragment, gene or allele.
  • the terms are used interchangeably and the genetic element, locus, introgression fragment, gene or allele can thus be transferred into any other genetic background lacking the trait. Not only seeds deposited and comprising the genetic element, locus, introgression fragment, gene or allele can be used, but also progeny/descendants from such seeds which have been selected to retain the genetic element, locus, introgression fragment, gene or allele, can be used and are encompassed herein, such as commercial varieties developed from the deposited seeds or from descendants thereof.
  • Whether a plant comprises the same genetic element, locus, introgression fragment, gene or allele as obtainable from the deposited seeds can be determined by the skilled person using one or more techniques known in the art, such as phenotypic assays, whole genome sequencing, molecular marker analysis, trait mapping, chromosome painting, allelism tests and the like.
  • the invention provides a spinach plant comprising resistance against at least Peronospora farinosa races 7-14, wherein said resistance is conferred by a single gene.
  • the gene designated herein RPF13 (for Resistance to Peronospora Farinosa 13) further optionally confers resistance against one or more or all of Peronospora farinosa races 1 -6, or at least against Pfs 1 -2 and Pfs 4-6 (it is understood that when Pfs races 1-6, or one or more or all of races 1-6, are referred to elsewhere herein that in one aspect race 3 is excluded, i.e. only resistance against Pfs races 1-2 and 4-6 is referred to).
  • the RPF13 gene therefore, confers resistance against all currently known pathogenic Pfs races, races 1-14, when in homozygous or heterozygous form in the plant.
  • the RPF13 gene confers resistance against pathogenic Pfs races, races 1-2 and 4-14, when in homozygous or heterozygous form in the plant.
  • the resistance gene also confers resistance against one or more pathogenic isolates of Pfs, such as isolate UA4712 or other isolates, including future isolates or races which develop in the field due to large scale uses of Pfs resistant spinach varieties.
  • the RPF13 gene confers resistance against at least pathogenic Pfs races 7-14 and against UA4712, when in homozygous or heterozygous form in the plant.
  • the RPF13 gene is a single, dominant resistance gene, i.e. when a plant comprising RPF13 in homozygous form (such as a plant grown from seed deposited under accession number NCIMB 42160) is crossed with a susceptible plant, such as variety Viroflay, the F2 progeny will segregate in a 3 (resistant) : 1 (susceptible) ratio.
  • a single, dominant resistance gene which is effective against all know pathogenic races, is of great advantage in generating resistant spinach varieties.
  • the Fl hybrid variety Andromeda is a stack of two resistance genes, one inherited from the female parent and one from the male parent line.
  • a representative sample of seeds of a spinach line comprising the RPF13 gene in homozygous form has been deposited under Accession number NCIMB 42160.
  • a spinach plant comprising the RPF13 resistance gene is obtainable by (or obtained by, or derivable from, or derived from) crossing a spinach plant grown from seeds deposited under accession number NCIMB 42160, with another spinach plant, for example with a spinach plant lacking Pfs resistance genes (a susceptible plant) or with a spinach plant comprising one or more different Pfs resistance genes.
  • a spinach plant comprising resistance against at least Peronospora farinose races 7-14 is provided wherein the resistance is conferred by a single dominant gene, called RPF13, wherein the RPF13 gene is the gene as present in seeds deposited under NCIMB42160 or progeny thereof.
  • the RPF13 gene is, therefore the gene as found in plants (or plant parts) grown from seeds deposited under accession number NCIMB 42160 or in progeny of such plants, such as plants obtained by selfing NCIMB 42160 or by crossing NCIMB 42160 with another spinach plant to obtain progeny and by retaining the resistance gene in the progeny (e.g. using phenotypic and/or molecular methods to identify or select progeny containing the RPF13 gene).
  • a spinach plant comprising resistance against Peronospora farinosa races 7-14, wherein said resistance is conferred by a single gene, wherein said gene is the gene as found in (or as obtainable from) seeds deposited under accession number
  • NCIMB42160 e.g. by crossing a plant grown from seeds of accession number NCIMB42160 with another spinach plant.
  • the (dominant) gene is in homozygous or heterozygous form.
  • the RPF13 gene (and the Pfs resistance phenotype conferred by the gene), can be transferred from the seeds deposited under NCIMB 42160, or from progeny of said seeds, into any spinach line or variety by traditional breeding techniques and can confer race 7-14 resistance, optionally further race 1- 6 resistance and/or UA4712 resistance (and optionally resistance against new pathogenic isolates) onto another spinach plant.
  • a spinach plant of the invention can be used as male or female parent in a cross with another spinach plant, and progeny, such as Fl, F2, F3, or further generations of selling and/or backcross progeny (e.g. BC1, BC2, BC1 S1, BC2S1, BC1 S2, etc.) can be identified and selected, whereby the progeny comprise the same Pfs resistance phenotype as the initial plant of the invention.
  • progeny such as Fl, F2, F3, or further generations of selling and/or backcross progeny (e.g. BC1, BC2, BC1 S1, BC2S1, BC1 S2, etc.)
  • progeny such as Fl, F2, F3, or further generations of selling and/or backcross progeny (e.g. BC1, BC2, BC1 S1, BC2S1, BC1 S2, etc.)
  • progeny such as Fl, F2, F3, or further generations of selling and/or backcross progeny (e.g. BC1, BC2, BC1 S1, BC2S1, BC1 S2, etc.
  • a spinach comprising resistance against at least Peronospora farinosa races 7-14, optionally further comprising resistance against races 1-6, and/or optionally further comprising resistance against isolate UA4712 and optionally new pathogenic isolates.
  • the spinach plant comprises resistance against at least Pfs races 7-14.
  • the spinach plant comprises resistance against at least Pfs races 1-6 and 7-14 (i.e. Pfs 1-14).
  • the spinach plant comprises resistance against at least Pfs races 7-14 and UA4712 and optionally further new pathogenic isolates.
  • the spinach plant comprises resistance against at least Pfs races 1-6, 7-14 and UA4712 and optionally further new pathogenic isolates.
  • a spinach plant genotype i.e. a spinach line or variety
  • comprises resistance against one or more Pfs races or isolates can be tested using qualitative disease resistance assays under controlled environment conditions. Different protocols of such assays exist and can be used by the person skilled in the art.
  • seedlings of a plurality of plants of the plant genotype to be tested e.g. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
  • seedlings are inoculated with inoculum of the Pfs race and the seedlings are incubated under conditions which are favorable to the pathogen.
  • the plants are assessed for infection symptoms, especially sporulation on the cotyledons and/or leaves (e.g.
  • each plant is categorized as "resistant” (showing no signs of sporulation) or "susceptible” (showing sporulation). If a certain percentage of all plants of a genotype are classified as “resistant”, e.g. more than about 85%, 90%, 95%, 98%, 99% (or even 100%), then the spinach plant genotype is resistant to the race tested.
  • one or more control plants e.g. a susceptible line or variety, a resistant line or variety
  • selection or identification of a spinach plant comprising the RPF13 gene of the invention may be achieved by detecting one or more molecular markers linked to the RPF13 gene or locus. This aspect will be described elsewhere herein.
  • the spinach plant is an inbred line, especially an inbred line which can be used as a parent for Fl hybrid seed production.
  • the spinach plant is a hybrid, especially an Fl hybrid.
  • An Fl hybrid may be generated by crossing a first inbred parent line which comprises the RPF13 gene, preferably in homozygous form, with a second inbred parent line.
  • the first inbred parent line may be a line developed from using seeds deposited under NCIMB 42160 or from progeny of plants grown from these seeds, whereby the progeny retain the Pfs resistance phenotype (and the RPF13 gene).
  • the second inbred parent line may be any spinach line, i.e. it may completely lack Pfs resistance, or it may comprise a different Pfs resistance gene (and different resistance phenotype) or it may also comprise the RPF13 gene.
  • the spinach plant according to the invention may be any type of spinach, as the RPF13 gene can be easily transferred into any spinach line or variety.
  • the spinach plant may be a savoy type, a semi-savoy type or flat- or smooth leaved spinach.
  • the RPF13 gene can be introduced into any other spinach plant by introgression from a plant grown from seeds of which a representative sample was deposited under NCIMB 42160, or any spinach plant derived therefrom and comprising the RPF13 gene.
  • the deposited seeds are therefore a source of the RPF13 resistance gene of the invention, as are spinach plants not directly obtained from the deposit, but for example indirectly obtained (e.g. later released commercial varieties) and which contain the RPF13 gene of the invention.
  • RPF13 gene may be identified, e.g. in wild spinach or wild relatives of spinach and e.g. an allelism test may be used to determine whether another dominant gene, conferring the same Pfs resistance phenotype as the plant of the invention (or as progeny thereof), is the same gene or a different gene.
  • allelism test may be used to determine whether another dominant gene, conferring the same Pfs resistance phenotype as the plant of the invention (or as progeny thereof), is the same gene or a different gene.
  • Alternative methods to determine whether another gene is the same gene include the development of molecular markers linked to the RPF13 gene of the invention and analyzing whether the markers also occur in plants comprising the other gene.
  • the RPF13 gene was identified in wild material from a genebank and was introduced through backcrossing into S. oleracea.
  • a spinach plant comprising resistance against at least Pfs races 7-14, optionally further against 1-6, and/or optionally further against UA4712 and/or new pathogenic isolates, wherein said resistance against Peronospora farinosa is conferred by an introgression fragment from wild spinach or from a wild relative of spinach, wherein the wild relative is selected from Spinacia tetranda and Spinacia turkestanica.
  • the introgression fragment is the fragment as found in (and as obtainable from; or obtained from; or derivable from; or derived from) spinach seeds, a representative sample of seeds having been deposited under accession number NCIMB 42160.
  • the fragment can be identified by various methods, such as chromosome painting or sequencing the spinach genome and identifying chromosome parts which are introgressions from wild spinach or wild relatives of spinach.
  • the fragment can also be identified by one or more molecular markers (e.g. SNP markers, AFLP markers, RFLP markers, etc.), especially molecular markers which are polymorphic between cultivated spinach and the wild introgression fragment.
  • molecular markers e.g. SNP markers, AFLP markers, RFLP markers, etc.
  • the plant comprising the RPF13 gene as found in NCIMB 42160 comprises the same RPF13 markers or genomic gene sequence or introgression fragment as found in NCIMB42160 (or in progeny thereof) .
  • the introgression fragment is derived from the fragment as found in spinach seeds, a representative sample of seeds having been deposited under accession number NCIMB 42160, whereby the introgression fragment is shorter but retains the RPF13 gene (and the Pfs resistance phenotype conferred by the gene).
  • Spinach plants comprising such shorter introgression fragments can be generated by crossing a plant of the invention with another spinach plant and selecting recombinant progeny which retain the resistance phenotype conferred by the RPF13 gene, but which contain a shorter introgression fragment.
  • a method for generating a spinach plant comprising resistance against at least Pfs races 7-14, optionally further against one or more or all of races 1-6, and/or optionally against UA4712 comprising the steps of: a) Providing a spinach plant comprising resistance against at least Pfs races 7-14, optionally further against one or more or all of races 1-6, and/or optionally against UA4712; b) Crossing said spinach plant with another spinach plant to produce Fl seeds; c) Optionally selfing the plants grown from Fl seeds one or more times to produce F2, F3 or
  • further generation selfing progeny d) Identifying (or selecting) spinach plants grown from Fl, F2, F3 or further generation selfing progeny which have resistance against at least Pfs races 7-14, optionally further against one or more or all of races 1-6, and/or optionally against UA4712; e) Optionally crossing said identified (or selected) Fl progeny or selfing progeny to the spinach plant of step b), to produce a backcross progeny; f) Optionally selecting backcross progeny comprising resistance against at least Pfs races 7-14, optionally further against one or more or all of races 1-6, and/or optionally against UA4712.
  • a method for generating a spinach plant comprising resistance against at least Peronospora farinosa races 7-14 comprises the steps of: a) Providing a spinach plant comprising an introgression fragment obtainable from (or as in)
  • accession NCIMB 42160 which introgression fragment confers resistance against at least Peronospora farinosa races 7-14 (optionally further against one or more or all of races 1-6, and/or optionally against UA4712); b) Crossing said spinach plant with another spinach plant, for example with a spinach plant which is susceptible against one or more of Peronospora farinosa races 7-14, to produce Fl seeds; c) Optionally selling the plants grown from Fl seeds one or more times to produce F2, F3 or
  • further generation selling progeny d) Identifying spinach plants grown from Fl, F2, F3 or further generation selfing progeny which have resistance against at least Peronospora farinosa races 7-14 (optionally further against races 1-6, and/or optionally against UA4712) and/or which comprise the introgression fragment or a resistance-conferring part of the introgression fragment; e) Optionally crossing said identified Fl progeny or selfing progeny to the spinach plant of step b), to produce a backcross progeny; f) Optionally selecting backcross progeny which comprises resistance against at least Peronospora farinosa races 7-14 (optionally further against one or more or all of races 1-6, and/or optionally against UA4712) and/or which comprise the introgression fragment or a resistance-conferring part of the introgression fragment.
  • the plant of a) comprises the RPF13 gene as found in seeds deposited under accession number NCIMB 42160.
  • the spinach plant may be the plant grown from the seeds of the deposit or any spinach plant made using, or having used, the seed deposit and which retains the Pfs resistance phenotype (and the gene conferring it). This includes commercial spinach varieties which were made using the seed deposit.
  • the spinach plant of a) comprises the RPF13 gene according to the invention, e.g. as found in (or as obtainable from; obtained from; derivable from; derived from) NCIMB 42160.
  • the plant in a) may therefore be a plant grown from seeds, a representative sample of which has been deposited under NCIMB42160 (comprising RPF13 in homozygous form) or from progeny of such seeds which retain the RPF13 gene and phenotype.
  • Selections (or identification) in step d) and/or f) may be made based on the phenotype (i.e. using a Pfs resistance assay) and/or based on molecular methods, such as detection of molecular markers linked to the RPF13 gene or locus, or other methods such as sequencing.
  • the spinach plant of step (a) preferably comprises the RPF13 gene (i.e. the mtrogression fragment comprising the RPF13 gene) in homozygous form.
  • step b) the spinach plant is, in one aspect, crossed with a spinach plant which is susceptible against at least one of the Pfs races against which the plant of a) is resistant. If the second parent in b) is a spinach plant which is susceptible against at least one of the Pfs races against which the plant of a) is resistant, then the selection in step (d) and/or (f) may be based on selecting plants which now have resistance against that race.
  • Steps e) and f) may be repeated one or more times.
  • plants can be selected and/or identified which retain the Pfs resistance phenotype conferred by the RPF13 gene, but which have a smaller introgression fragment.
  • This can have advantages, as negative traits coupled to the wild introgression fragment can thereby be removed.
  • Initial introgession fragments from wild sources can be quite large, e.g. 20Mb or 30Mb. It is therefore preferred to reduce the size of the introgression fragment by recombination and to select plants comprising smaller introgression fragments, but which retain the resistance-conferring part.
  • spinach with all sizes of introgression fragments originating from (or derived from; or derivable from; or obtained from; or obtainable from) seeds deposited under accession number NCIMB 42160 are included herein, as long as the Pfs resistance conferring part (i.e. the RPF13 gene) is retained in the spinach plant.
  • the presence can be tested / selected phenotypically and/or using molecular methods known in the art.
  • the plants according to the invention may be any cultivated spinach, e.g. savoy, semi-savoy, flat- or smooth leaved spinach. They may be inbred lines, Fl hybrids, double haploids, transgenic plants, mutant plants, a single locus converted plant comprising the RPF13 gene, etc.
  • Plants of the invention can be used to generate progeny, which have or retain the Pfs resistance phenotype as obtainable from (as present in; as derivable from; as obtained or derived from) seeds deposited under NCIMB 42160.
  • a spinach according to the invention can be selfed and/or crossed one or more times with another spinach plant and seeds can be collected.
  • the presence of the RPF13 gene in the progeny plants can be determined (i.e. progeny plants comprising the RPF13 gene can be identified/selected) by the Pfs resistance phenotype and/or molecular methods, such as molecular markers (e.g. SNP markers) closely linked to the RPF13 gene or locus.
  • molecular markers e.g. SNP markers
  • a spinach plant of which representative seeds have been deposited under accession number NCIMB 42160, or progeny thereof (e.g. obtained by selfing), for generating a spinach plant comprising Pfs resistance against at least Peronospora farinosa races 7-14 (optionally further against races 1-6, and/or optionally against UA4712) is provided.
  • a spinach plant comprising resistance against at least Peronospora farinosa races 7-14 conferred by an introgression fragment obtainable from (or as present in; as derivable from; as obtained or derived from) seeds deposited under accession number NCIMB 42160, or from progeny thereof (e.g. obtained by selfing), for generating spinach plant comprising resistance against at least Peronospora farinosa races 7-14 (optionally further against races 1-6, and/or optionally against UA4712) is provided.
  • allelism tests can be used to determine whether the resistance gene in a spinach plant is the same gene or a different gene as the RPF13 gene as present in NCIMB42160 (or in progeny thereof). So, NCIMB42160 (or progeny) can be crossed with another spinach plant comprising the same resistance phenotype and in progeny of such a cross one can determine in which ratios the phenotype segregates. So in one aspect a spinach plant is provided comprising resistance against P. ferinosa races 7-14, wherein said resistance gene conferring said resistance phenotype is the dominant RPF13 gene as present in NCIMB42160 (or progeny thereof), i.e.
  • allelic to RPF13 is a different allele of the RPF13 gene found in NCIMB42160, as determinable in an allelism test. Allelism tests for dominant genes are known in the art and are e.g. described in Hibberd et al. 1987 (Phytopathology 77: 1304-1307).
  • seeds from which any of the plants of the invention can be grown are provided, as are containers or packages containing or comprising such seeds. Seeds can be distinguished from other seeds due to the presence of the RPF13 resistance gene, either phenotypically (based on plants having the RPF13 resistance phenotype) and/or using molecular methods.
  • seeds are packaged into small and/or large containers (e.g., bags, cartons, cans, etc.).
  • the seeds may be pelleted prior to packing (to form pills or pellets) and/or treated with various compounds, such as seed coatings.
  • Seed pelleting can be combined with film coating (Halmer, P. 2000. Commercial seed treatment technology. In: Seed technology and its biological basis. Eds: Black, M. and Bewley, J. D., pages 257- 286). Pelleting creates round or rounded shapes, which are easily sown with modern sowing machines.
  • a pelleting mixture typically contains seeds and at least glue and filler material. The latter could be, for example, clay, mica, chalk or cellulose.
  • certain additives can be included to improve particular properties of the pellet, e.g., a seed treatment formulation comprising at least one insecticidal, acaricidal, nematicidal or fungicidal compound can be added directly into the pelleting mixture or in separate layers.
  • a seed treatment formulation can include one of these types of compounds only, a mixture of two or more of the same type of compounds or a mixture of one or more of the same type of compounds with at least one other insecticide, acaricide, nematicide or fungicide.
  • Formulations especially suitable for the application as a seed treatment can be added to the seed in the form of a film coating including also the possibility of using the coating in or on a pellet, as well as including the seed treatment formulation directly into the pellet mixture.
  • a film coating is a uniform, dust- free, water permeable film, evenly covering the surface of all individual seeds (Halmer, P. 2000. Commercial seed treatment technology. In: Seed technology and its biological basis. Eds: Black, M. and Bewley, J.
  • the coating mixture generally also contains other ingredients such as water, glue (typically a polymer), filler materials, pigments and certain additives to improve particular properties of the coating.
  • glue typically a polymer
  • filler materials typically a polymer
  • pigments typically a pigment
  • certain additives to improve particular properties of the coating.
  • the film coating can be added on the outside of the pellet, in between two layers of pelleting material, and directly on the seed before the pelleting material is added. Also more than 1 film coating layer can be incorporated in a single pellet.
  • a special type of pelleting is encrusting. This technique uses less filler material, and the result is a 'mini- pellet'.
  • Seeds may also be primed. Spinach is often primed. Priming is a water-based process that is performed on seeds to increase uniformity of germination and emergence from the soil, and thus enhance vegetable stand establishment. Priming decreases the time span between the emergence of the first and the last seedlings. Methods how to prime spinach seeds are well known in the art (see, e.g., Chen et al. 2010, Seed Sci. & Technol. 38: 45-57).
  • Plant parts and vegetative reproductions In a further aspect plant parts, obtained from (obtainable from) a plant of the invention are provided herein, and containers or packages comprising said plant parts.
  • the plant parts are leaves of spinach plants of the invention, preferably harvested leaves, or parts of these.
  • Leaves may be loose, bunched, fresh (e.g. in bags), frozen, blanched or boiled. Leaves may be fresh or processed, they may be part of food or feed products, such as salads, etc.
  • seeds include for example seeds produced on the plant of the invention after selfpollination or seed produced after cross-pollination, e.g. pollination of a plant of the invention with pollen from another spinach plant or pollination of another spinach plant with pollen of a plant of the invention.
  • the plant part is a plant cell.
  • the plant part is a non- regenerable cell or a regenerable cell.
  • the plant cell is a somatic cell.
  • a non-regenerable cell is a cell which cannot be regenerated into a whole plant through in vitro culture, but the non-regenerable cell may be in a plant or plant part (e.g. leaves) of the invention.
  • the plant cell is a reproductive cell, such as an ovule or pollen. These cells are haploid. When they are regenerated into whole plants, they comprise the haploid genome of the starting plant. If chromosome doubling occurs (e.g. through chemical treatment), a double haploid plant can be regenerated.
  • the plant of the invention, comprising the RPF13 resistance gene is a haploid or a double haploid spinach plant.
  • in vitro cell culture or tissue culture of spinach plants of the invention in which the cell- or tissue culture is derived from a plant parts described above, such as, for example and without limitation, leaves, pollen, embryos, cotyledon, hypocotyls, callus, meristematic cells, roots, root tips, anthers, flowers, seeds or stems, somatic cells, reproductive cells.
  • spinach plants regenerated from the above-described plant parts, or regenerated from the above-described cell or tissue cultures, said regenerated plant having a Pfs resistance phenotype (as conferred by the RPF13 gene), i.e. retains the RPF13 gene (or the introgression fragment comprising the RPF13 gene) of the invention.
  • Pfs resistance phenotype conferred by the RPF13 gene
  • These plants can also be referred to as vegetative propagations of plants of the invention.
  • harvested leaves of plants of the invention and packages comprising a plurality of leaves of plants of the invention. These leaves thus comprise the RPF13 gene of the invention, detectable by e.g. linked molecular markers or phenotypically (for the originally used whole plant and/or regenerated plant).
  • the invention also provides for a food or feed product comprising or consisting of a plant part described herein.
  • the food or feed product may be fresh or processed, e.g., canned, steamed, boiled, fried, blanched and/or frozen etc. Examples are salad or salad mixtures comprising leaves or parts of leaves of plants of the invention.
  • a spinach plant of the invention or a progeny thereof retaining the Pfs resistance phenotype conferred by the RPF13 gene and/or retaining the introgression fragment comprising the RPF13 gene, as present in NCIMB 42160, and parts of the afore-mentioned plants, can be suitably packed for, e.g., transport, and/or sold fresh.
  • Such parts encompass any cells, tissues and organs obtainable from the seedlings or plants, such as but not limited to: leaves, cuttings, pollen, parts of leaves, and the like.
  • Leaves may be harvested immature, as baby-leaf or baby spinach, or mature.
  • a plant, plants or parts thereof may be packed in a container (e.g., bags, cartons, cans, etc.) alone or together with other plants or materials. Parts can be stored and/or processed further.
  • a container e.g., bags, cartons, cans, etc.
  • Parts can be stored and/or processed further.
  • food or feed products comprising one or more of such parts, such leaves or parts thereof obtainable from a plant of the invention, a progeny thereof and parts of the afore-mentioned plants.
  • containers such as cans, boxes, crates, bags, cartons, Modified Atmosphere Packaging, films (e.g. biodegradable films), etc. comprising plant parts of plants (fresh and/or processed) of the invention are also provided herein.
  • plants and parts of spinach plants of the invention, and progeny of spinach plants of the invention are provided, e.g., grown from seeds, produced by sexual or vegetative reproduction, regenerated from the above-described plant parts, or regenerated from cell or tissue culture, in which the reproduced (seed propagated or vegetatively propagated) plant comprises resistance against at least Pfs races 7-14, optionally further against races 1-6, and/or optionally against UA4712 (as conferred by the RPF13 gene).
  • a plant, progeny or vegetative propagation comprises the Pfs resistance phenotype as conferred by the RPF13 gene can be tested phenotypically using e.g. the Pfs disease resistance assays as described above or in the Examples; and/or using molecular techniques such as molecular marker analysis, DNA sequencing (e.g. whole genome sequencing to identify the wild introgression), chromosome painting, etc.
  • the RPF13 resistance gene obtainable from (obtained from; as found in) plants deposited under NCIMB 42160, or progeny thereof can be combined with other Peronospora farinosa resistance genes or resistance loci (e.g. RPF1- RPF6, R6, etc.) or with other traits, such resistance against bacteria (e.g. Pseudomonas syringae pv. spinacea; Erwinia carotovora), fungi (e.g. Albugo occidentalis; Colletotrichum dematium f. sp. spinaciae; Stemphylium botryosum f. sp. spinacia), viruses (e.g.
  • viruses causing curly top disease or nematodes.
  • This can be done by traditional breeding techniques, e.g. by backcrossing in order to introduce one or more traits into a plant of the invention or in order to introduce the RPF13 gene of a plant of the invention into another spinach plant comprising such one or more additional traits.
  • a plant of the invention is used as a donor of the RPF13 gene, while in another aspect a plant of the invention is used as recipient of one or more other traits.
  • the invention provides for progeny comprising or retaining the Pfs resistance phenotype (conferred by the RPF13 gene), such as progeny obtained by, e.g., selling one or more times and/or cross-pollinating a plant of the invention with another spinach plant of a different variety or breeding line, or with a spinach plant of the invention one or more times.
  • the invention provides for progeny that retain the RPF13 gene (conferring the Pfs resistance phenotype) of (as found in) NCIMB 42160.
  • the invention provides for a progeny plant comprising the RPF13 resistance, such as a progeny plant that is produced from a spinach plant comprising the RPF13 resistance by one or more methods selected from the group consisting of: selfing, crossing, mutation, double haploid production or transformation.
  • Mutation may be spontaneous mutations or human induced mutations or somaclonal mutations.
  • plants or seeds of the invention may also be mutated (by e.g. irradiation, chemical mutagenesis, heat treatment, TILLING, etc.) and/or mutated seeds or plants may be selected (e.g. natural variants, somaclonal variants, etc.) in order to change one or more characteristics of the plants.
  • plants of the invention may be transformed and regenerated, whereby one or more chimeric genes are introduced into the plants.
  • Transformation can be carried out using standard methods, such as Agrobacterium tumefaciens mediated transformation or biolistics, followed by selection of the transformed cells and regeneration into plants.
  • a desired trait e.g. genes conferring pest or disease resistance, herbicide, fungicide or insecticide tolerance, etc.
  • a desired trait can be introduced into the plants, or progeny thereof, by transforming a plant of the invention or progeny thereof with a transgene that confers the desired trait, wherein the transformed plant retains the RPF13 gene and the Pfs resistance phenotype conferred by it and contains the desired trait.
  • the RPF13 gene or allele may be transferred to progeny by further breeding.
  • progeny are Fi progeny obtained by crossing a plant of the invention with another plant or SI progeny obtained by selfing a plant of the invention. Also encompassed are F2 progeny obtained by selfing the Fi plants, or further generation progeny. "Further breeding” encompasses traditional breeding techniques (e.g., selfing, crossing, backcrossing), marker assisted breeding, and/or mutation breeding.
  • the progeny have the Pfs resistance phenotype of NCIMB 42160 and comprising the RPF13 gene (or introgression comprising the gene or the reduced size introgression fragment comprising the RPF13 gene) obtainable / obtained from (or as found in) NCIMB 42160.
  • haploid plants and/or double haploid plants of plant of the invention are encompassed herein, which comprise resistance against at least Peronospora farinosa races 7-14 (optionally further against races 1-6, and/or optionally against UA4712), as conferred by the RPF13 gene or by the introgression fragment comprising the RPF13 gene.
  • Haploid and double haploid (DH) plants can for example be produced by anther or microspore culture and regeneration into a whole plant. For DH production chromosome doubling may be induced using known methods, such as colchicine treatment or the like. So, in one aspect a spinach plant is provided, comprising Pfs resistance phenotype as described, wherein the plant is a double haploid plant.
  • the invention in another embodiment relates to a method for producing spinach seed, comprising crossing a plant of the invention with itself or a different spinach plant and harvesting the resulting seed.
  • the invention relates to seed produced according to this method and/or a spinach plant produced by growing such seed.
  • a plant of the invention may be used as male and/or female parent, in the production of spinach seeds, whereby the plants grown from said seeds comprise at least Peronospora farinosa races 7-14 (optionally further against races 1-6, and/or optionally against UA4712), due to the presence of the RPF13 gene.
  • progeny of a spinach plant of the invention are provided, wherein the progeny plant is produced by selling, crossing, mutation, double haploid production or transformation and wherein the progeny retain the RPF13 resistance gene (and phenotype conferred by it) described herein, i.e.
  • the resistance gene or locus (or introgression fragment comprising the gene or locus) as present in / found in / as derived from (or as derivable from) seed deposit NCIMB 42160 is retained in the progeny plants.
  • Molecular markers may also be used to aid in the identification of the plants (or plant parts or nucleic acids obtained therefrom) containing the RPF13 resistance gene or locus or allele(s). For example, one can develop one or more suitable molecular markers which are closely genetically (and preferably also physically) linked to the RPF13 resistance gene, locus or allele.
  • the segregating population can then be phenotyped for Pfs resistance and genotyped using e.g.
  • molecular markers such as SNPs (Single Nucleotide Polymorphisms), AFLPs (Amplified Fragment Length Polymorphisms; see, e.g., EP 534 858), or others, and by software analysis molecular markers which co-segregate with the Pfs resistance trait in the segregating population can be identified and their order and genetic distance (centimorgan distance, cM) to the RPF13 resistance gene or locus can be identified.
  • Molecular markers which are closely linked to RPF13 resistance locus e.g. markers at a 5 cM distance or less, can then be used in detecting and/or selecting plants (e.g.
  • plants of the invention or progeny of a plant of the invention or plant parts comprising or retaining the introgression fragment comprising the RPF13 resistance gene or locus.
  • Such closely linked molecular markers can replace phenotypic selection (or be used in addition to phenotypic selection) in breeding programs, i.e. in Marker Assisted Selection (MAS).
  • MAS Marker Assisted Selection
  • flanking markers are used in MAS, i.e. one marker on either side of the RPF13 gene or locus.
  • Any other type of molecular marker and/or other assay that is able to identify the relative presence or absence of a trait of interest (i.e. the RPF13 gene or locus) in a plant or plant part can also be useful for breeding purposes.
  • EXAMPLE 1 Pfs resistance phenotype of spinach comprising the RPF13 gene
  • the resistance to downy mildew infection was assayed with the help of a differential set obtained from the Naktuinbouw.
  • the inoculated plants were covered with transparent plastic material with 100% relative humidity for a 24 h period, after this period the plastic was removed on top to lower the relative humidity to 80%. After 10 days, the plants were scored as 'resistant' or 'susceptible' based on symptoms of pathogen sporulation on the cotyledons and true leaves, as described by Irish et al. (2007; Plant Dis . 91 : 1392- 1396). Plants exhibiting any evidence of sporulation were considered 'susceptible'. Plants not exhibiting sporulation were considered 'resistant'. Resistant plants were re- inoculated to assess whether plants initially scored as resistant had escaped infection, or whether they were truly resistant. These plants were scored again 10 days after the second inoculation.
  • the resistance of a spinach hybrid is the effect of two genes, as described by Handke et al. (2000; Schmbaustatt . 65: 73-78).
  • the RPF2 resistance gene (Lazio type, resistance against Pfs races) is combined with a RPF3 (Califlay type, resistance against Pfs races 1, 3, 5, 8, 9, 11, 12, and 14) resistance gene.
  • This combination of genes results in resistance to Pfs races 1-12, and 14 and susceptibility to Pfs race 13 (see Table 2).
  • the new resistance gene, RPF13 gives full resistance to all currently known races. The resistance works both homozygous in fixed lines as in heterozygous backgrounds, like spinach hybrids (see Table 3).
  • the RPF13 resistance trait of the present invention is conferred by a single dominant resistance gene, which has the great advantage that the RPF13 resistance trait can be easily transferred into other spinach lines or varieties by crossing/introgression, and that it can easily be combined with other resistance genes.
  • the RPF13 gene confers full resistance to current Pfs races without the need to select a complimentary resistance gene.
  • Table 2 shows the differential set of spinach downy mildew races and the resistance of various spinach varieties (hybrids) to each one of these pathogenic races.
  • a susceptible reaction (indicated as “+” in the table) is scored when a successful infection by the fungus is visible as sporulation on the cotyledon or leaf.
  • Resistance (indicated as “-” in the table) is the absence of sporulation on the cotyledons and mature leaves.
  • “+/-” indicates variability in the number of resistant and susceptible plants observed, 'n is not tested.
  • *In WO2013/064436 the R6 gene is described as conferring resistance against Pfs 1-6, Pfs9, Pfsl 1, Pfs 12, Pfs 13 and Pfs 14 (UA4410).
  • EXAMPLE 2 Introduction of the RPF13 resistance trait into other spinach plants
  • a plant of the invention is crossed (as a mother) with a different spinach plant susceptible to all known races. Plants of the Fl population were selfed, and plants of the F2 generation are tested for Pfs resistance, as described in Example 1.
  • Table 3 gives a detailed overview of the segregation of the RPF12 resistance gene in a number of Pfs assays. Chi-square tests confirmed that the observed segregation in the F2 populations was consistent with a 3:1 (resistant : susceptible) segregation of the RPF13 resistance profile.
  • Table 3 Overview of segregation of the RPF13 resistance profile in eight Pfs assays, expressed in the number and percentage of plants observed as resistant or susceptible per Pfs race. The plant belong to the progeny of an inbred from a cross between a spinach plant which lacks the RPF13 gene and a genotype of the invention containing the RPF13 gene. Chi-square tests confirm that the results fit the expected segregation pattern for a dominant monogenic trait. In all cases chi-square values were well above 0.05.

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Abstract

La présente invention concerne le domaine de la culture des épinards, en particulier un nouveau gène de résistance dominante, appelé RPF13, qui confère une résistance vis-à-vis de toutes les races de Peronospora farinosa et elle concerne des épinards comprenant ledit gène.
PCT/EP2014/069130 2013-09-13 2014-09-09 Épinards résistants au mildiou WO2015036378A1 (fr)

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US20150240256A1 (en) * 2014-02-27 2015-08-27 Seminis Vegetable Seeds, Inc. Compositions and methods for peronospora resistance in spinach
WO2017081187A1 (fr) * 2015-11-10 2017-05-18 Pop Vriend Research B.V. Résistance au peronospora dans spinacia sp
WO2017081189A1 (fr) * 2015-11-10 2017-05-18 Pop Vriend Research B.V. Résistance au peronospora dans spinacia sp
WO2018060474A1 (fr) * 2016-09-30 2018-04-05 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Procédé de modification du profil de résistance de spinacia oleracea au mildiou
US9974276B2 (en) * 2015-11-20 2018-05-22 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Peronospora resistance in Spinacia oleracea
US10039258B2 (en) 2013-10-08 2018-08-07 Seminis Vegetable Seeds, Inc. Methods and compositions for Peronospora resistance in spinach
CN108697058A (zh) * 2015-11-20 2018-10-23 瑞克斯旺种苗集团公司 菠菜中的霜霉属抗性
CN109104855A (zh) * 2016-05-13 2018-12-28 瑞克斯旺种苗集团公司 菠菜中非r基因介导的抗性
WO2019145446A1 (fr) 2018-01-26 2019-08-01 Nunhems B.V. Plants d'épinard résistants au moins aux races 8 et 10 à 16 de peronospora farinosa
WO2019145447A1 (fr) 2018-01-26 2019-08-01 Nunhems B.V. Plants d'épinard résistants au moins aux races 8, 9, 11, 13 et 16 de peronospora farinosa
WO2019233863A1 (fr) 2018-06-04 2019-12-12 Bayer Aktiengesellschaft Benzoylpyrazoles bicycliques utilisés comme herbicide
US10633670B2 (en) * 2017-09-29 2020-04-28 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Method for modifying the resistance profile of spinacia oleracea to downy mildew
US20210282345A1 (en) * 2020-03-12 2021-09-16 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Peronospora resistance in spinacia oleracea
US11202421B2 (en) 2016-09-30 2021-12-21 Rijk Zwaan Zaadteelt En Zaadhandek B.V. Peronospora resistance in spinacia oleracea
US11203766B2 (en) 2016-09-30 2021-12-21 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Peronospora resistance in spinacia oleracea
US11277983B2 (en) 2015-11-10 2022-03-22 Pop Vriend Seeds Bv Peronospora resistance in spinacia sp
WO2022090546A1 (fr) * 2020-10-30 2022-05-05 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Résistance aux peronospora dans spinacia oleracea
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US11473102B2 (en) 2020-10-30 2022-10-18 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Peronospora resistance in Spinacia oleracea
WO2022239824A1 (fr) 2021-05-12 2022-11-17 株式会社サカタのタネ Plant d'épinard ayant un nouveau gène résistant au mildiou
US11603538B2 (en) 2018-12-21 2023-03-14 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Peronospora resistance in Spinacia oleracea
WO2023074911A1 (fr) 2021-11-01 2023-05-04 株式会社サカタのタネ Épinard possédant un nouveau gène de résistance au mildiou
CN116507200A (zh) * 2020-10-30 2023-07-28 瑞克斯旺种苗集团公司 菠菜中的霜霉病抗性

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JP2021511063A (ja) * 2018-01-26 2021-05-06 ヌンヘムス、ベスローテン、フェンノートシャップＮｕｎｈｅｍｓ Ｂ．Ｖ． 少なくともＰｅｒｏｎｏｓｐｏｒａ ｆａｒｉｎｏｓａのレース８、９、１１、１３および１６に耐性のホウレンソウ植物
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