SPINACH PLANTS RESISTANT TO AT LEAST PERONOSPORA FARINOSA RACES 8 AND 10 TO 16

Abstract

The invention relates to a cultivated spinach plant having a new resistance against at least Peronospora farinosa races 8 and 10 to 16, seed, cell cultures and progeny of said plant, use of the plant with the resistance, and methods for generating and identifying such a plant.

Claims

1. A spinach plant of the species Spinacia oleracea comprising an introgression fragment from a donor that is a wild relative of spinach, wherein said introgression fragment comprises a single gene, which confers resistance against at least Peronospora farinosa races 8 to 16 when the gene is in homozygous form, and said gene is linked to SEQ ID NO: 1 comprising an Adenine at nucleotide 114 (SNP_01), or to a sequence comprising at least 90% sequence identity to SEQ ID NO: 1 and comprising an Adenine at the nucleotide position equivalent to nucleotide 114 of SEQ ID NO: 1.

2. The plant according to claim 1, wherein said single gene confers resistance against at least Peronospora farinosa races 1, 2 and 6 to 17 when the gene is in homozygous form.

3. The plant according to claim 1, wherein said resistance gene does not confer resistance against Peronospora farinosa races 3, 4 and 5.

4. The plant according to claim 1, wherein said resistance gene is the gene as found in spinach seeds having been deposited under accession number NCIMB 42607 or progeny thereof.

5. The plant according to claim 1, wherein said donor is of the species Spinacia turkestanica.

6. The plant according to claim 5, wherein said introgression fragment is the fragment as found in spinach seeds having been deposited under accession number NCIMB 42607, or a sub-fragment of said introgression fragment retaining said resistance gene and retaining SEQ ID NO: 1.

7. The plant according to claim 1, wherein said resistance gene is obtainable by crossing a spinach plant grown from seeds deposited under accession number NCIMB 42607, with another spinach plant.

8. The plant according to claim 1, wherein said spinach plant is a hybrid plant, and said hybrid plant comprises said resistance gene in heterozygous form or homozygous form, or wherein said spinach plant is an inbred plant or a male parent line or a female parent line comprising said resistance gene in homozygous form.

9. The plant according to claim 1, wherein the spinach plant is savoy, semi-savoy, flat- or smooth leaved or oriental.

10. Seed from which a plant according to claim 1 can be grown.

11. A progeny plant of a spinach plant according to claim 1, wherein said progeny plant retains the resistance gene and retains SNP_01 comprising an Adenine at nucleotide 114 of SEQ ID NO: 1, or an Adenine at the equivalent position in a sequence comprising at least 90% sequence identity to SEQ ID NO: 1.

12. The progeny plant according to claim 11, wherein the progeny plant is produced by one or more methods selected from the group consisting of: selfing, crossing, double haploid production or transformation.

13. A method for generating a spinach plant comprising resistance against at least Peronospora farinosa races 8 and 10 to 16, comprising crossing a spinach plant comprising an introgression fragment obtainable from seed as deposited under NCIMB 42607, wherein the introgression fragment comprises SEQ ID NO: 1, with another spinach plant and optionally selfing the progeny plant of step a one or more times to produce a further generation selfing progeny and optionally collecting seeds produced on the plant.

14. A part of a spinach plant according to claim 1, wherein the part is a leaf, a part of a leaf, a stem, a part of a stem, a stalk, a part of a stalk, a shoot, a part of a shoot, a bud or a part of a bud, a cutting, a root, a part of a root, a root tip, a petiole, a part of a petiole, a cotyledon, a part of a cotyledon, a flower, a part of a flower, a petal, a part of a petal, a stamen, a part of a stamen, an anther, a part of an anther, pollen, a stigma, a part of a stigma, a style, a part of a style, an ovary, a part of an ovary, an ovule, a part of an ovule, a seed, a part of a seed, a seed coat, an embryo, a part of an embryo, a hypocotyl, an embryo sac, a fruit, a part of a fruit, a cell, a protoplast, callus, a microspore, meristem, or cambium, wherein said plant part retains the resistance gene conferring resistance against at least Peronospora farinosa races 8 to 16 when the gene is in homozygous form and retaining SNP_01 comprising an Adenine at nucleotide 114 of SEQ ID NO: 1, or an Adenine at the equivalent position in a sequence comprising at least 90% sequence identity to SEQ ID NO: 1.

15. A cell culture or tissue culture comprising at least one cell or a tissue of a spinach plant according to claim 1, wherein said cell culture or tissue culture retains the resistance gene conferring resistance against at least Peronospora farinosa races 8 to 16 when the gene is in homozygous form and further retains SNP_01 comprising an Adenine at nucleotide 114 of SEQ ID NO: 1, or an Adenine at the equivalent position in a sequence comprising at least 90% sequence identity to SEQ ID NO: 1.

16. A spinach plant regenerated from the cell culture or tissue culture of claim 15, wherein said plant retains the resistance gene conferring resistance against at least Peronospora farinosa races 8 to 16 when the gene is in homozygous form and further retains SNP_01 comprising an Adenine at nucleotide 114 of SEQ ID NO: 1, or an Adenine at the equivalent position in a sequence comprising at least 90% sequence identity to SEQ ID NO: 1.

17. A method for identifying or selecting a spinach plant comprising an introgression fragment from a donor that is a wild relative of spinach, wherein said introgression fragment comprises a single gene, which confers resistance against at least Peronospora farinosa races 8 to 16 when the gene is in homozygous form, said method comprising: determining the presence of an Adenine at nucleotide 114 of SEQ ID NO: 1 (SNP_01), or of an Adenine at the nucleotide position equivalent to nucleotide 114 of SEQ ID NO: 1 in a sequence comprising at least 90% sequence identity to SEQ ID NO: 1. in a spinach plant.

18. A cell of a cultivated spinach plant comprising an introgression fragment from a donor that is a wild relative of spinach, wherein said introgression fragment comprises a single gene, which confers resistance against at least Peronospora farinosa races 8 to 16 when the gene is in homozygous form, and said gene is linked to SEQ ID NO: 1 comprising an Adenine at nucleotide 114 (SNP_01), or to a sequence comprising at least 90% sequence identity to SEQ ID NO: 1 and comprising an Adenine at the nucleotide position equivalent to nucleotide 114 of SEQ ID NO: 1.

19. (canceled)

20. A method for generating a spinach plant that confers resistance in heterozygous and homozygous form, against at least Peronospora farinosa races 8 to 16, comprising crossing a spinach plant comprising an introgression fragment from a donor that is a wild relative of spinach, with another spinach plant to produce a progeny plant; and optionally selfing the progeny plant of step a one or more times to produce a further generation selfing progeny and optionally producing seed, wherein said introgression fragment comprises a single gene, which confers resistance against at least Peronospora farinosa races 8 to 16 when the gene is in homozygous form, and said gene is linked to SEQ ID NO: 1 comprising an Adenine at nucleotide 114 (SNP_01), or to a sequence comprising at least 90% sequence identity to SEQ ID NO: 1 and comprising an Adenine at the nucleotide position equivalent to nucleotide 114 of SEQ ID NO: 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0120] Plants and Methods of the Invention

[0121] In one embodiment, the invention provides a cultivated spinach plant having resistance against at least Peronospora farinosa races 8 and 10 to 16, preferably against at least races 8 and 10 to 17, where the resistance is conferred by a single dominant gene.

[0122] The single gene is designated RPF14, for Resistance to Peronospora farinosa gene 14. Thus the invention provides RPF14 which confers dominant resistance against Peronospora farinosa races 8 and 10 to 16, preferably 8 and 10 to 17. In another embodiment, RPF14further confers resistance against Peronospora farinosa races 1, 2, 6, 7 and 9, at least when the RPF14 gene is in homozygous form. In a further aspect of the invention, RPF14 confers resistance to isolate UA0514 and/or potentially other pathogenic isolates of Peronospora farinosa. These other isolates potentially comprise future isolates which develop in the field. The gene does not confer resistance to races 3, 4 and 5. The RPF14gene was identified in a wild relative of spinach and was introduced through backcrossing into Spinacia oleracea, preferably cultivated spinach. The RPF14 gene is a single gene. The gene inherits dominantly for at least the resistance to Pfs races 8 and 10 to 16, preferably 8 and 10 to 17; that is, when a plant comprising RPF14 in homozygous form is crossed with a susceptible plant, such as variety Viroflay, the F1 progeny will all show resistance to at least Pfs races 8 and 10 to 16, preferably to races 8 and 10 to 17, and in the F2 progeny said resistance will segregate in a 3 (resistant): 1 (susceptible) ratio. The RPF14 gene is present in homozygous form in seed deposited under accession number NCIMB 42607, i.e. the introgression fragment comprising RPF14 is present in homozygous form. The RPF14gene is linked to the resistant donor nucleotide SNP_01, which comprises an Adenine at nucleotide 114 of SEQ ID NO: 1 or Adenine at the equivalent position in a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Thus, the introgression fragment present in the deposited seeds comprises SEQ ID NO:1, i.e. comprises an Adenine at nucleotide 114 of SEQ ID NO: 1, and the Adenine (and the SEQ ID N:1) is present in homozygous form in the deposited seeds (the SNP_01 genotype of the deposited seeds is AA).

[0123] In an aspect of the invention the RPF14 gene from S turkestanica confers resistance against at least Pfs races 8 and 10 to 16, preferably against races 8 and 10 to 17, when the gene is in homozygous or in heterozygous form in the genome of a cultivated spinach plant of the species S. oleracea.

[0124] In a further aspect of the invention the RPF14gene from S. turkestanica confers resistance against at least Pfs races 8 to 17, or at least races 6 to 17, or at least races 1, 2 and 6 to 17, at least when the gene is in homozygous form, and to several of those races also in heterozygous form, in the genome of a cultivated spinach plant of the species S. oleracea.

[0125] In another aspect of the invention RPF14confers resistance to at least races 8 and 10-16, preferably 8 and 10 to 17, and further to one or more of Pfs races selected from races 1, 2, 6, 7 and 9, at least when RPF14 (or the introgression fragment comprising RPF14) is in homozygous form, and to several of those races also when the gene (or introgression fragment comprising the gene) is in heterozygous form, in the genome of a cultivated spinach plant.

[0126] In further aspects of the invention RPF14 confers resistance to resistance to at least races 8 and 10-16, preferably at least 8 and 10 to 17, when in homozygous or heterozygous form and further to Pfs race 1 when RPF14 (or the introgression fragment comprising RPF14) is in homozygous or in heterozygous form, and/or to Pfs race 2 when RPF14 (or the introgression fragment comprising RPF14) is in homozygous or in heterozygous form, and/or to Pfs race 6 when RPF14 (or the introgression fragment comprising RPF14) is in homozygous or in heterozygous form, and/or to Pfs race 7 when RPF14 (or the introgression fragment comprising RPF14) is in homozygous or in heterozygous form and/or to Pfs race 9 when RPF14 (or the introgression fragment comprising RPF14) is in homozygous or in heterozygous form in the genome of a cultivated spinach plant. In yet a further aspect of the invention RPF14 confers resistance to isolate UA0514 and/or another pathogenic Pfs isolate when RPF14 (or the introgression fragment comprising RPF14) is in homozygous or in heterozygous form in the genome of a cultivated spinach plant.

[0127] In a further aspect of the invention, RPF14 introgression confers resistance to at least Pfs races 8 and 10-16, preferable 8 and 10 to 17 in a cultivated spinach plant, wherein the RPF14gene (or the introgression fragment comprising the gene) is linked to (comprises) the resistant donor nucleotide of SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position in a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Resistance against races 8 and 10 to 16, or 8 and 10 to 17, is conferred at least when the introgression fragment is in homozygous form or in heterozygous form, as the resistance to these races is dominant. For the resistance against Pfs 1, 2, 6, 7, 9 and UA0514, it is not clear if the resistance is only seen when the RPF14 gene is in homozygous form or if it is also seen (for one or more of these races) when the RPF14 gene is in heterozygous form, this is depending on whether resistance against a race is dominant or recessive. Whether resistance against a race is dominant or recessive can be tested in a resistance assay in e.g. plants heterozygous for RPF14 and/or segregating for RPF14.

[0128] In a further aspect of the invention, RPF14 introgression fragment confers resistance to at least Pfs races 7 to 16, 7 to 17, or 8 to 17, in a cultivated spinach plant, wherein the RPF14 gene (or the introgression fragment comprising the gene) is linked to (comprises) the resistant donor nucleotide of SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position in a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Resistance against these races is conferred at least when the introgression fragment is in homozygous form, optionally also when the introgression fragment is in heterozygous form, depending on whether resistance against a race is dominant or recessive. Whether resistance against a race is dominant or recessive can be tested in a resistance assay in e.g. plants heterozygous for RPF14 and/or segregating for RPF14.

[0129] In yet a further aspect of the invention, RPFNintrogression confers resistance to at least Pfs races 6 to 16, or 6 to 17, in a cultivated spinach plant, wherein the RPF14 gene (or the introgression fragment comprising the gene) is linked to (comprises) the resistance donor nucleotide of SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position in a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Resistance against these races is conferred at least when the introgression fragment is in homozygous form, optionally also when the introgression fragment is in heterozygous form, depending on whether resistance against a race is dominant or recessive. Whether resistance against a race is dominant or recessive can be tested in a resistance assay in e.g. plants heterozygous for RPF14 and/or segregating for RPF14.

[0130] Resistance against Pfs races 8 and 10-16 and 17 was found to be conferred in a dominant manner. Whether RPF14 confers resistance against races 1, 2, 6, 7, 9 and/or UA0514 in a dominant or in a recessive manner has to be determined. As mentioned, the skilled person can easily determine this. What is known is that, when RPF14 (or the introgression fragment comprising RPF14) is present in homozygous form, the cultivated spinach plant is resistant against these races. In the deposited seeds the introgression fragment is present in homozygous form. So plants grown from said seeds can be crossed with a plant lacking the RPF14 gene, to generate F1 plants, and the F1 and/or F2 and/or F3 population can be tested for resistance to each of the Pfs races, in order to determine if the resistance conferred is seen when RPF14 is in heterozygous form (dominant) or only when RPF14 is in homozygous form (recessive).

[0131] A representative sample of seeds of a cultivated spinach line comprising the RPF14gene (that is the introgression fragment comprising the RPF14 gene) in homozygous form has been deposited under the Budapest Treaty under Accession number 42607 by Nunhems B.V. on 12 Jul. 2016, at NCIMB Ltd. Thus, in an embodiment of the invention, the RPF14 resistance gene is the gene found in seed deposited under accession number NCIMB 42607, or from a plant ora part thereof grown from seed deposited under accession number NCIMB 42607, or from a cell culture derived from said seed or said plant or said part thereof. Obviously, also progeny of NCIMB 42607 are encompassed, which progeny comprise the RPF14 gene in their nuclear genome.

[0132] When referring herein to a cultivated spinach plant or plant part comprising the RPF14 gene this is understood to mean that the spinach plant or plant part comprises the introgression fragment, which fragment comprises the RPF14 gene from a wild S. turkestanica donor at the RPF14 locus on the chromosome. In one aspect the wild S. turkestanica donor is the same donor as in the deposited seeds, i.e. the S. turkestanica sequence of the RPF14 gene and of the fragment comprising the RPF14 gene has the same nucleotide sequence as in the deposited seeds. This can be determined by e.g. whole genome sequencing. Alternatively, the wild S. turkestanica donor may be a different accession, comprising the RPF14 gene (conferring e.g. the same Pfs resistance) but having a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the RPF14 gene of the deposited seeds or to the introgression fragment comprising the RPF14 gene of the deposited seeds.

[0133] The cultivated spinach line of which a representative sample of seeds were deposited under NCIMB 42607, comprising the S. turkestanica introgression fragment carrying RPF14 in homozygous form, is resistant against Pfs races 1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and UA0514.

[0134] The RPF14 gene is located on an introgression fragment from a wild relative of spinach. In one aspect of the invention, the introgression fragment is from Spinacia turkestanica and comprises in addition to the RPF14 gene a molecular marker linked to the RPF14 gene and which can be used to select a fragment comprising RPF14. An Adenine at nucleotide 114 of SEQ ID NO: 1 (the resistant donor nucleotide for SNP_01) was found to be linked to the RPF14 gene on the introgression fragment. Susceptible lines lacking the introgression fragment were found to contain either a Guanine at nucleotide 114 of SEQ ID NO: 1 (as shown in SEQ ID NO: 2) or a Guanine at nucleotide 120 of SEQ ID NO: 3, which nucleotide is the equivalent nucleotide to nucleotide 114 of SEQ ID NO: 1 or 2, as can be seen from the pairwise alignment (using the Emboss program Needle). The sequence of the susceptible S. oleracea plant lines may thus show variation at the SNP marker region. Thus, in one aspect the RPF14 gene is linked to an Adenine at nucleotide 114 of SEQ ID NO: 1 or to an Adenine at the equivalent position in a sequence comprising at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. In an aspect of the invention, the resistance gene, RPF14, is obtained or obtainable from an S. turkestanica accession which accession has the same Pfs resistance phenotype as conferred by RPF14 (e.g. as the deposited seeds) and comprises an Adenine at nucleotide 114 of SEQ ID NO: 1 (the resistant donor nucleotide for SNP_01) or an Adenine at the equivalent position in a sequence comprising at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0135] In another aspect of the invention, the introgression fragment comprising RPF14 is the introgression as present in (and as obtainable from; or obtained from; or derivable from; or derived from) a spinach seed deposited under accession number NCIMB 42607 or a subfragment thereof (retaining RPF14), wherein said introgression fragment (or subfragment) comprises the RPF14 gene conferring resistance to at least Pfs races 8 and 10 to 16. In one aspect the introgression fragment comprises also SEQ ID NO: 1.

[0136] The introgression fragment present in the deposited seeds is from a specific donor accession and thus has a unique nucleotide sequence. The whole fragment can be easily transferred into other spinach lines or varieties, by crossing a plant grown from the deposited seeds with another spinach plant and selecting a descendant comprising the introgression fragment. Selection can be by various methods, by the Pfs resistance phenotype and/or selecting progeny comprising SEQ ID NO: 1 and/or sequencing, SNP genotyping (selecting progeny comprising an Adenine for SNP_01, etc.).

[0137] 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 introgression fragment from the wild donor. Typically, a mapping population is used to generate markers. For example markers which are specific for the introgression fragment may be generated which are within 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM from the RPF14 gene and/or within 1 Mb, 0.9 Mb, 0.8 Mb, 0.7 Mb, 0.6 Mb, 0.5Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb, or less from the RPF14 gene. In a particularly preferred embodiment the introgression fragment comprising the RPF14 gene is obtained through a method that comprises the step of growing a seed of NCIMB 42607 into a plant.

[0138] In another embodiment a cultivated spinach plant is provided which comprises the RPF14 gene on a sub-fragment of the introgression fragment present in the seed of NCIMB 42607. Such plants can be generated by selfing or crossing a plant grown from seed of NCIMB 42607 with another spinach plant and selecting descendants which have a shorter introgression fragment, i.e. where a recombination event occurred between homologous chromosomes within the introgression fragment, so that part of the fragment is recombined off. For example recombinant inbred lines can be generated which have different sub-fragments of the original full-size introgression fragment present in seeds of NCIMB 42607. The original introgression fragment from the S turkestanica donor is estimated to be equal to or less than 3.0 Mb in size, especially equal to or less than 2.0 Mb in size. Sub-fragments comprising RPF14 may thus be less than 3.0 Mb, less than 2.0 Mb, such as less than 1.0 Mb, 0.7 Mb, 0.6 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb or less and may still comprise the RPF14 gene. Optionally sub-fragments also retain SEQ ID NO: 1.

[0139] As mentioned previously, in the mapping population for RPF14, the SNP nucleotide of the SNP_01 from the S. turkistanica donor is an Adenine at position 114 of SEQ ID NO: 1, instead of Guanine, which is the SNP nucleotide of the recurrent parent (S. oleracea, lacking the introgression), as shown at position 114 of SEQ ID NO: 2 or at the position 120 of SEQ ID NO: 3 (position 120 of SEQ ID NO: 3 is the equivalent position to position 114 of SEQ ID NO: 1 or SEQ ID NO: 2). SEQ ID NO: 2 and SEQ ID NO: 3 are found in susceptible lines (see also FIG. 1 of the Examples).

[0140] A diploid spinach plant homozygous for the introgression fragment comprising RPF14 therefore has, an Adenine at the SNP_01 position of each of the homologous chromosomes (i.e. AA genotype). A spinach plant heterozygous for the introgression fragment has an Adenine at the SNP_01 position of one chromosome, and a Guanine, Cytosine or Thymine at the equivalent position of the other chromosome, depending on the recurrent parent background (i.e. AG or AC or AT genotype).

[0141] The present invention encompasses introgression fragments from the S. turkestanica donor as present in the deposited seeds (comprising RPF14 and optionally comprising SEQ ID NO: 1; and an Adenine at nucleotide 114 of SEQ ID NO:1) and also from other S. turkestanica donors comprising RPF14 on the same chromosome locus on chromosome 3 of the spinach genome, but wherein the introgression fragment has a nucleotide sequence which is not 100% identical to the sequence of the introgression fragment present in the deposited seeds (e.g. having only at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity to the introgression fragment present in the deposited seeds). Such an introgression fragment may in one aspect comprise SNP_01, wherein SNP_01 has an Adenine at a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1. Thus the marker sequence of SEQ ID NO: 1 may also not be 100% identical in such a different S. turkestanica donor. The invention further also encompasses sub-fragments of such introgression fragments from other S. turkestanica donors, comprising RPF14 and optionally comprising an Adenine at nucleotide 114 of a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0142] Thus, the invention also encompasses a sub-fragment of aforementioned introgression fragment comprising the RPF14 gene, wherein said sub-fragment comprises the RPF14 gene conferring resistance to at least Pfs races 8 and 10 to 16, preferably Pfs 8 and 10 to 17, and is a part of the introgression fragment as present in seed deposited under accession number NCIMB 42607 or is a part of an introgression fragment of a different S. turkestanica donor having substantial sequence identity to the introgression fragment present in the deposited seeds. The invention encompasses said sub-fragment comprising the RPF14 gene and comprising the resistant donor nucleotide for SNP_01, which is an Adenine at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position in a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Thus, the introgression sub-fragment is in one aspect obtained (and as obtainable from; or derivable; or derived) from the fragment as found in cultivated spinach seeds as deposited under accession number NCIMB 42607, and the sub-fragment retains the RPF14 gene (and the Pfs resistance phenotype conferred by the gene, and optionally SEQ ID NO: 1), and the introgression fragment is in another aspect obtained from another S. turkestanica donor comprising an RPF14 gene at the same locus on chromosome 3. Spinach plants comprising such a shorter introgression fragment can be generated by crossing a plant of the invention with another spinach plant and selecting a recombinant progeny which retains the resistance phenotype conferred by the RPF14 gene, but which comprise a shorter introgression fragment. The skilled person can, for example, cross a plant grown from the deposited seeds with another cultivated spinach plant (e.g. a plant susceptible to one or more of Pfs races 8 and 10 to 17), and then self the F1 progeny to produce an F2 population and identify recombinants (cross-over events) having occurred in the introgression fragment.

[0143] As mentioned previously, WO2015054339 describes a QTL on chromosome 6. The locus was introgressed from S. tetrandra and confers broad spectrum Pfs resistance, in particular resistance to races 7, 10, 11, 12, 13, and 14 of Peronospora farinosa f. sp. spinathe (Pfs), or to races 1-14 and UA4712 of Peronospora farinosa f. sp. spinathe (Pfs). (UA4712 is Pfs race 15). Chromosome 6 actually corresponds to chromosome 3 in SpinachBase. The two S. tetrandra sequences which flank the QTL, herein provided as SEQ ID NO: 4 and 5, are located at 1.4 Mb (SEQ ID NO: 4) and 0.7 Mb (SEQ ID NO: 5). The instant SNP_01 marker from S. turkestanica is, on the other hand located at 0.6 Mb of the chromosome 3.

[0144] Inventors have also tested whether the sequences, which flank the QTL in S. tetrandra, are present in the seeds deposited under accession number NCIMB 42607. Neither the left nor the right flanking sequences (i.e. SEQ ID NO: 4 and 5) were present in the deposited seeds, as further described in the Examples. Instead, S. oleracea DNA was present in the deposited seeds at the corresponding chromosome region (provided as SEQ ID NO: 6 and 7).

[0145] Thus, in one aspect, a cultivated spinach plant of the invention, which comprises an introgression comprising RPF14, does not comprise the broad spectrum resistance locus described in WO2015054339. Of course, as this locus is in a different region of chromosome 3, it can be combined with RPF14 without undue burden, even with both RPF14 and the QTL in homozygous form.

[0146] The introgression fragment of the invention comprising the RPF14 gene, as present in seed deposited under NCIMB 42607, does not comprise SEQ ID NO: 4 or SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5 are linked to the resistance-conferring introgression from S. tetrandra described in WO2015054339. SEQ ID NO: 4 and SEQ ID NO: 5 are not present in the introgression fragment of the invention, or in seed of the invention as deposited under NCIMB 42607. The seed deposited under NCIMB 42607, which comprise the RPF14 gene comprises SEQ ID NO: 6 at the equivalent region to SEQ ID NO: 4. The seed deposited under NCIMB 42607, which comprise the RPF14gene comprises SEQ ID NO: 7 at the equivalent region to SEQ ID NO: 5.

[0147] The RPF14gene is useful because it is a single gene which confers dominant resistance to several pathogenic Peronospora farinosa races, that is at least Pfs races 8 and 10 to16, preferably at least races 8 and 10 to 17. RPF14 can be used in generating resistant spinach varieties. In the art, resistance genes are commonly stacked (combined with other complementary resistance genes) to provide resistance against a large number of Peronospora farinosa races. To stack resistance genes in a hybrid variety, the gene should confer dominant resistance. This is especially important for conferring Peronospora farinosa resistance in the diploid spinach, because some resistance genes are allelic, limiting the number of possible combinations. Therefore, the products described herein (e.g. plants, plant parts, progeny plants, etc.) provide a significant improvement over the prior art.

[0148] In one aspect, the invention provides a spinach F1 hybrid plant and plant parts (and seed from which an F1 hybrid can be grown), wherein one parent is an inbred line which comprises the RPF14 gene of the invention in homozygous form. The other parent may be susceptible, or it may be an inbred parent line comprising a P. farinosa resistance gene selected from the group RPF1, RPF2, RPF3, RPF4, RPF5, RPF6, RPF7, RPF8, RPF9, RPF11, RPF12, RPF15, the R6 gene (of WO2013/064436), the p10 gene (of WO2017/194073), the R15 gene (of WO2017/084724) or the gene described in US20170127641 or US20170127642.

[0149] Also a method for producing a hybrid spinach seed is provided, comprising crossing a first parent spinach plant with a second parent spinach plant and harvesting the resulting hybrid spinach seed, wherein the first parent spinach plant comprises the RPF14 gene conferring dominant resistance to at least Pfs races 8 and 10 to 16, preferably to 8 and 10 to 17, and that requires stacking with another downy mildew resistance gene to have resistance against races 3, 4 and/or 5; and/or to have resistance against races 1, 2, 6, 7 and/or 9. Thus, in one aspect the other parent is an inbred parent line comprising a P. farinose resistance gene selected from the group RPF1, RPF2, RPF3, RPF4, RPF5, RPF6, RPF7, RPF8, RPF9, RPF11, RPF12, the R6 gene (of WO2013/064436), the p10 gene (of WO2017/194073), the R15 gene (of WO2017/084724) or the gene described in US20170127641 or US20170127642. Also encompassed is a F1 hybrid spinach seed, and a hybrid spinach plant or plant part grown from such seed, produced by this method.

[0150] For providing additional resistance against races 3, 4 and/or 5, the following genes are most suitable: RPF1, RPF2, RPF3, RPF4, RPF6, RPF7, RPF8, RPF9, RPF11, RPF12, R6, R15 and the gene described in US20170127641 or US20170127642.

[0151] For providing additional resistance against races 1, 2, 6, 7 and 9, the following genes are most suitable: RPF1, RPF2, RPF8, RPF9, RPF11 and RPF12 and the gene described in US20170127641 or US20170127642. Thus in a preferred aspect, the F1 hybrid plant (or seed from which the plant can be grown) comprises the RPF14 gene from one parent and a gene selected from RPF1, RPF2, RPF8, RPF9, RPF11, RPF12, the gene described in US20170127641 or in US20170127642 from the other parent.

[0152] The combination of RPF14 gene from one parent with the following gene from the other parent provides a spinach plant which is resistance against Pfs 1 to Pfs 17: RPF1, RPF2, RPF8, RPF9, RPF12, the gene described in US20170127641 or in US20170127642.In one aspect of the invention a spinach plant comprising the RPF14 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 42607, 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. An example of a suitable susceptible plant is variety Viroflay.

[0153] The spinach plant of the invention may e.g. be an inbred line, comprising RPF14 in homozygous form, or an F1 hybrid comprising the RPF14 gene in either homozygous or heterozygous form.

[0154] In one embodiment, the RPF14 resistance gene of the invention can be combined with other Peronospora farinosa resistance genes or resistance loci (e.g. RPF7- RPF9, RPF7? or RPF72, R6, R75, or the resistances disclosed in WO2015054339 and EP2912940 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, Stemphyllum botryosum f. sp. spinacia), viruses (e.g. a virus causing Curly top disease, or Speckles, or Spinach blight, or Spinach Mosaic) or nematodes (e.g. Clover cyst nematode (Heterodera trifolli), Lesion nematode (Pratylenchus spp.), Root-knot nematode (Meloidogyne spp.) or Sugarbeet cyst nematode (Heterodera schachtii)). Combining can, for example, 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 RPF14 gene of a plant of the invention into another spinach plant comprising such one or more additional traits or by other techniques, including gene editing or transformation. In one aspect a plant of the invention is used as a donor of the RPF14gene, while in another aspect a plant of the invention is used as recipient of one or more other traits. A skilled person can obtain a hybrid plant resistant to all currently known Pfs races, namely Pfs 1 to 16, or 1 to 17, by combining the RPF14 gene with other suitable resistance genes. For example, RPF14 can be combined with RPF72 or RPF1, or RPF2, or RPF8, or RPF9, or the gene described in US20170127641 or US20170127642, to obtain resistance to all currently known Pfs races.

[0155] The RPF14resistance gene, or the introgression fragment on which it is located, or a sub-fragment of the fragment comprising RPFN can be transferred from a plant of the invention to another spinach plant by various methods known to the skilled person. A donor of the RPF14 resistance gene may thus be e.g. a plant grown from the deposited seeds, or a progeny plant thereof.

[0156] Thus a donor of the RPF14 resistance gene can be NCIMB 42607 or a progeny of a plant grown from said deposit, a progeny of said plant, or a plant grown from a cell culture derived from said plant. The transferred RPF14 gene can confer resistance to at least Pfs races 8 and 10 to 16, preferably races 8 and 10 to 17, and also resistance to one or more of Pfs races 1, 2, 6, 7 and 9 and resistance to Pfs isolate UA0514 in the recipient plant.

[0157] The RPF14resistance gene, or the introgression fragment on which it is located, or a sub-fragment thereof comprising the gene, can be used to make a hybrid plant (e.g. an F1 hybrid), or an inbred plant or a homozygous plant, optionally a doubled haploid plant. In a further aspect, the inbred or homozygous plant is a male parent line, preferably a male elite parent. In a yet further aspect, the inbred or homozygous plant is a female parent line, preferably a female elite parent. A male parent line can be crossed with a female parent line to make F1 hybrid seeds, comprising RPF14 (or the introgression fragment comprising RPF14, and optionally comprising SEQ ID NO: 1) in homozygous form.

[0158] In an embodiment, the parent line functions as a donor of the RPF14 resistance gene. Said donor plant can be crossed with another spinach plant, and progeny can be obtained, including F1, F2, F3, or further generation selfing progeny, backcross progeny (e.g. BC1, BC2, BC1S1, BC2S1, BC1S2, etc.) etc. Plants having the same Pfs resistance phenotype as the initial plant of the invention can be identified and selected among the progeny. Likewise, the introgression fragment can be detected in the progeny, e.g. by detecting markers indicative of the introgression fragment (e.g. SNP_01) or sequencing, etc.

[0159] In one aspect, the inbred line is a cultivated plant of the species Spinacia oleracea comprising resistance against at least Peronospora farinosa races 8 and 10 to 16 (preferably at least races 8 and 10 to 17), wherein said resistance is conferred by the single gene introgressed from Spinacia turkestanica (RPF14), said gene being linked to the resistant donor nucleotide for SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably at least 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0160] The RPF14 resistance gene, or the introgression fragment on which it is located, or a sub-fragment thereof, can also be transferred in various types of spinach, such as: savoy, semi-savoy, flat- or smooth leaved or oriental spinach. Preferably, the savoy, semi-savoy, flat- or smooth leaved or oriental cultivated spinach plant is a hybrid plant.

[0161] In one embodiment, a cultivated spinach plant is encompassed comprising resistance against Pfs races 8 and 10 to 16, preferably at least against races 8 and 10 to 17, wherein said resistance is conferred by a single dominant gene RPF14 introgressed from a wild relative of spinach, preferably S. turkestanica, which gene is linked to SEQ ID NO: 1 or to a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably at least 97%, 98% or 99% sequence identity to SEQ ID NO: 1. In one aspect the nucleotide at position 114 (or the equivalent nucleotide in a pairwise alignment) of any of these sequences is an Adenine. The RPF14 gene can be identified in different accessions of wild relatives of spinach, especially in accessions of the species S. turkestanica, and can be introgressed into cultivated spinach. To do so, the skilled person can e.g. screen such an accession for the presence of the resistant donor nucleotide of SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably at least 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and/or test the Pfs resistance phenotype and optionally the inheritance (as single gene) to determine if that accession contains RPF14. Optionally also sequencing, fine mapping, allelism tests, etc. can be done to determine if the gene in the accession is indeed the RPF14 gene.

[0162] In a specific aspect, the resistance against Peronospora farinosa in a cultivated plant is conferred by an introgression fragment from Spinacia turkestanica. The cultivated spinach plant therefore comprises the RPF14 gene derived from S. turkestanica and is optionally linked to the resistant donor nucleotide of SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably at least 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0163] Presence of the resistance gene RPF14 may be determined by resistance tests for at least Pfs races 8 and 10 to 16 (preferable at least 8 and 10 to 17), optionally also resistance to one or more of Pfs races 1, 2, 6, 7 and 9 and/ or resistance to isolate UA0514 and/or other Pfs isolates. In an alternative embodiment resistance against a Pfs races, or a selection of Pfs races can be used as to indicate that the gene is transferred from a donor to a recipient plant. Thus, if e.g. the recipient parent in the cross lacks resistance against a particular Pfs race, then selection of a progeny plant which is resistant against that race indicates the transfer of the RPF14 gene.

[0164] The tests for presence of the resistance gene in a cultivated spinach plant (i.e. a spinach line or variety) comprise for example qualitative disease resistance assays under controlled environment conditions. The skilled person is familiar with applying different protocols for such assays. In short, 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 plants) are inoculated with inoculum of the Pfs race to be tested and the seedlings are incubated under conditions which are favorable to the pathogen. Several days after incubation, the plants are assessed for infection symptoms, especially sporulation on the cotyledons and/or leaves (e.g. first true leaf), and 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. Obviously, also one or more control plants (e.g. a susceptible line or variety, a resistant line or variety) should be included in the assay using the same treatment(s) and environmental conditions, to ensure that the assay works as expected.

[0165] Such a test for presence of RPF14 may be done on plants homozygous or heterozygous for the gene, using any isolate or race of Pfs. If the plant is categorized as resistant according to the test, when the gene is present in heterozygous form, the resistance is dominant. A simple test may comprise crossing a plant comprising the resistance gene with a plant which is susceptible to at least one Pfs race (i.e. has no background resistance) and testing F1 progeny for resistance to that Pfs race. If that F1 progeny is resistant to that Pfs race, it can be concluded the resistance is dominant. Such a test has resulted in the conclusion that RPF14 confers dominant resistance to Pfs races 8 and 10 to 16 and preferably 17. Another suitable test for dominant monogenic inheritance is crossing a plant comprising the resistance gene with a plant susceptible to all Pfs races, selfing the progeny from that cross to generate a F2 generation and observing segregation of resistance to Pfs races. If the segregation is a 3:1 ratio of resistant to susceptible plants the resistance is dominant monogenic. If the plant is categorized as resistant according to the test, only when the gene is present in homozygous form, the resistance inherited recessively.

[0166] Presence of the RPF14 resistance gene (or introgression fragment comprising the gene) in a spinach plant or plant part (e.g. a cell) may be also determined directly. The skilled person is aware of methods for screening, selecting or identifying a cultivated spinach plant (e.g. a progeny plant) or a part of a spinach plant, or a cell or a cell culture comprising RPF14 of the invention may be achieved by detecting one or more molecular markers linked to the RPF14gene or locus, such as SNP_01. Thus, in one aspect the introgression fragment comprising the RPF14 gene is detectable by presence of the resistant donor nucleotide of SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or Adenine at the equivalent position of a sequence comprising at least 90%, preferably at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: land/or any other molecular marker linked to RPF14 and/or the S. turkestanica introgression fragment comprising the RPF14 gene. Thus, the genome, especially chromosome 3 of the genome, comprising the introgression fragment can be distinguished by the skilled person from the genome, especially chromosome 3 of the genome lacking the introgression fragment and comprising S. oleracea genomic sequence instead.

[0167] The RPF14 gene is located at the beginning of chromosome 3, in the region starting at 0 Mb to 2.0 Mb of the chromosome, especially in the region starting at 0.4 Mb and ending at 1.5 Mb of chromosome 3 (as found in the SpinachBase genome). Thus, if this region is sequenced and comprises S. turkestanica sequence, optionally also the SNP_01 marker linked to the gene, and the plant comprises the resistance phenotype as conferred by the RPF14 gene, then the plant or plant part (e.g. cell) comprises the RPF14 gene of the invention.

[0168] In another aspect, the invention provides a cultivated spinach seed comprising RPF14 as part of the introgression fragment or a sub-fragment of the introgression fragment, as present in the deposit under accession number NCIMB 42607. The invention also provides a plurality of cultivated spinach seeds comprising RPF14, preferably in a container.

[0169] The invention further provides a cultivated spinach plant comprising an introgression fragment from a donor that is a wild relative of spinach conferring dominant resistance to Peronospora farinosa races 8 and 10 to 16, preferably 8 and 10 to 17, and resistance (potentially only when the fragment is in homozygous form) to Pfs races 1, 2, 6, 7 and 9 and to isolate UA0514. In one aspect of the invention, the fragment is introgressed from S. turkestanica. In another aspect of the invention, the introgression fragment is the introgression as present in seed deposited under accession number NCIMB 42607, or a short fragment of that fragment. The invention therefore also encompasses a cultivated spinach plant comprising a sub-fragment of aforementioned introgression fragment, wherein said sub-fragment confers dominant resistance to Pfs races 8 and 10 to 16, preferably to races 8 and 10 to 17, and resistance (potentially only when the fragment is in homozygous form) to Pfs races 1, 2, 6, 7 and 9 and to isolate UA0514 and/or other Pfs isolates. The invention further encompasses a cultivated spinach plant comprising said sub-fragment, wherein said sub-fragment is a part of the introgression fragment as present in seed deposited under NCIMB 42607. The shorter sub-fragment retains the RPF14 gene.

[0170] The cultivated spinach plant of the invention can be a hybrid plant, especially an F1 hybrid, or an inbred plant for example an inbred line which can be used as a parent for F1 hybrid seed production or a homozygous plant, optionally a doubled haploid plant.

[0171] The RPF14gene can be transferred into any spinach line or variety.

[0172] In other words, the RPF14gene 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 42607, or any spinach plant derived therefrom and retaining the RPF14gene. The deposited seeds are therefore a source of the RPF14 resistance gene of the invention, as is a spinach plant not directly obtained from the deposit, but indirectly obtained (e.g. through a later released commercial varieties) and which comprises RPF14gene of the invention.

[0173] Other sources of the RPF14 gene may be identified, e.g. in wild relatives of spinach (especially other S. turkestanica accessions which have the same Pfs resistance phenotype and/or comprise the marker linked to RPF14 provided herein (SNP_01) and e.g. an allelism test may be used to determine whether another gene, conferring the same Pfs resistance phenotype, is the same gene or a different gene. Likewise, sequencing may be used to confirm the presence of the RPF14 gene. Alternative methods to determine whether another gene is the same gene include the development of at least one molecular marker linked to the RPF14 gene of the invention and analyzing whether said marker occurs in plants comprising the other gene. An example of a suitable marker is the resistant donor nucleotide of SNP_01, which has an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, preferably at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0174] In one aspect a method is provided for generating a cultivated spinach plant comprising the RPF14 gene, comprising the steps of: [0175] a) Crossing a spinach plant comprising the RPF14 gene as described herein with another spinach plant to produce a progeny plant; [0176] b) Optionally selfing the progeny plant of step a one or more times to produce a further generation selfing progeny and optionally producing seed;

[0177] In an embodiment, the other spinach plant of step a) is susceptible to at least one of Pfs races 8 and 10 to 16 or 17. In a further embodiment, the other spinach plant of step a) is an inbred plant or a homozygous plant or a male parent line or a female parent line, or preferably an elite male parent line or an elite female parent line.

[0178] In another aspect the method comprising steps a) and optionally b) is provided, followed by [0179] c) Identifying the progeny plant of step a or b that comprises the RPF14 resistance gene by determining whether the progeny plant comprises resistance against at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17) and/or comprises an Adenine at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, preferably at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1; and/or which comprises an introgression fragment from S. turkestanica comprising the RPF14 gene. [0180] d) Optionally crossing the identified progeny plant of step c to another spinach plant to produce a progeny plant or progeny seed.

[0181] In another embodiment a method for generating a spinach plant comprising the RPF14 gene (conferring resistance to at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), optionally further resistance to one or more of Pfs races 1, 2, 6, 7 and 9 and/or to isolate UA0514 and/or other Pfs isolates) is provided, comprising the steps of: [0182] a) Crossing a spinach plant comprising an introgression fragment obtainable from (or as present in) seed as deposited under NCIMB 42607, which introgression fragment comprises SEQ ID NO: 1, with another spinach plant; [0183] b) Optionally selfing the progeny plant of step a one or more times to produce a further generation selfing progeny and optionally collecting seeds produced on the plant;

[0184] In an embodiment, the other spinach plant of step a) is susceptible to at least one of Pfs races 8 and 10 to 16 or 17. In a further embodiment, the other spinach plant of step a) is an inbred plant or a homozygous plant or a male parent line or a female parent line, or preferably an elite male parent line or an elite female parent line.

[0185] In another aspect the method comprising steps a) and optionally b) is provided, followed by [0186] c) Identifying the progeny plant of step a or b that comprises the RPF14 resistance gene by determining whether the progeny plant comprises resistance against at least Pfs races 8 and 10 to 16 and/or comprises SEQ ID NO: 1; [0187] d) Optionally crossing the identified progeny plant of step c to another spinach plant of to produce a progeny plant or seed.

[0188] Regarding both methods, the following is encompassed herein: In one aspect the plant of step a) comprises the RPF14 gene as found in seeds deposited under accession number NCIMB 42607. 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 RPF14 gene conferring it) and optionally which retains SEQ ID NO: 1. This includes commercial spinach varieties which were made using the seed deposit. Thus, the spinach plant of a) comprises the RPF14 gene according to the invention, e.g. as found in (or as obtainable from; obtained from; derivable from; derived from) NCIMB 42607.

[0189] Selections (or identification) in step c) 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 RPF14gene or locus, for example of the resistant donor nucleotide of SNP_01, which comprises Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or Adenine at the equivalent position of a sequence comprising at least 90%, preferably at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, or other methods such as sequencing.

[0190] In the methods above, the spinach plant of step (a) preferably comprises the RPF14gene (i.e. the introgression fragment or a sub-fragment thereof comprising the RPF14 gene) in homozygous form. In step a) the spinach plant comprising the resistance is, in one aspect, crossed with another 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.

[0191] In the above methods also plants can be selected and/or identified which retain the Pfs resistance phenotype conferred by the RPF14 gene, but which have a smaller introgression fragment than the one present in the deposited seeds. This can have advantages, as negative traits of S. turkestanica coupled to the introgression fragment can thereby be removed. 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 gene. So, in one aspect 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 42607 are included herein, as long as the Pfs resistance conferring part (i.e. the RPF14gene) is retained in the spinach plant. As mentioned, the presence can be tested/selected phenotypically and/or using molecular methods known in the art.

[0192] Also provided is a method for generating a spinach plant comprising dominant resistance against at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17) comprises the steps of:

[0193] a) crossing a first spinach plant of the species Spinacia oleracea with a second spinach plant which second spinach plant is susceptible against one or more of Pfs races 8 and 10 to 16 or 17, wherein the first a spinach plant comprising resistance against Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), and said resistance is conferred by a single gene introgressed from S. turkestanica, which gene is linked to the resistant donor nucleotide of SNP_01, which has an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1;

[0194] b) selfing a plant grown from progeny of said crossing one or more times to produce a further generation selfing progeny and/or backcrossing a plant grown from progeny of said crossing or grown from the further generation selfing progeny with a spinach plant is susceptible against one or more of Pfs races 8 and 10 to 16 or 17; and c) identifying a spinach plant among the progeny plants of step b) that comprises the a single gene of the first parent plant of step a).

[0195] In one aspect, the genotype of SNP_01 (which is linked to the RPF14 gene) is used to identify a plant in step c). The nucleotide of SNP_01 is Adenine, i.e. the donor nucleotide. Therefore in one aspect the plant comprises an introgression fragment which comprises the donor SNP_01 nucleotide.

[0196] A plant produced by the above method is also an embodiment of the invention.

[0197] Also, a method for screening, identifying or detecting the presence of the RPF14 gene as described herein in a spinach plant or plant part is provided, comprising: [0198] a) screening a cultivated spinach plant or plant part or DNA of such plant or plant part using a molecular marker assay which detects at least one SNP marker selected from the group consisting of: [0199] i) the resistant donor nucleotide of SNP_01, which has an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, preferably at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1; [0200] ii) another marker linked to the RPF14 gene or to the introgression fragment comprising the RPF14 gene; and optionally [0201] b) identifying or selecting a plant or plant part comprising the resistant donor SNP nucleotide for

[0202] SNP_01, which is an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, preferably at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1; and/or another marker linked to the RPF14 gene or to the introgression fragment comprising the RPF14 gene.

[0203] In yet another aspect, a method for detecting whether a cultivated spinach plant comprises an introgression fragment comprising the RPF14 gene as described herein, is provided, said method comprising: [0204] a) screening a plant or plant part (or DNA obtained from said plant or plant part) using a molecular marker assay which detects at least one SNP marker selected from the group consisting of: [0205] i) the resistant donor nucleotide for SNP_01, which has an Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, preferably at least 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1; and/or [0206] ii) another marker linked to the RPF14 gene or to the introgression fragment comprising the RPF14 gene.

[0207] Also a cultivated spinach plant or plant part derived from, obtained from, obtainable from or derivable from or identified or detected in any of the above methods are embodiments of the invention, said plant comprising resistance to at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17) conferred by RPF14 or said plant part comprising the RPF14 gene (or introgression fragment comprising the gene, and optionally the marker linked to the gene).

[0208] A plant of the invention can be used to generate a progeny, which has or retains the Pfs resistance gene of the invention as obtainable from (as present in; as derivable from; as obtained or derived from) seeds deposited under NCIMB 42607. To generate progeny, 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 RPF14 gene in the progeny plants can be determined (i.e. progeny plants comprising the RPF14 gene can be identified/selected) by the Pfs resistance phenotype and/or molecular methods, such as molecular markers (e.g. SNP markers) linked to the RPF14 gene or locus.

[0209] The invention further contemplates use of the RPF14 gene (and use of an introgression fragment comprising the gene) to confer resistance against at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), optionally further to races 1, 2, 6, 7, 9 and isolate UA0514.

[0210] In one embodiment, the use of a spinach plant, or progeny of said plant (e.g. obtained by selfing), or of a cell or cell culture that is regenerable, or a plant part that can be vegetatively propagated, is provided for generating a cultivated spinach plant comprising resistance to at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), optionally further to one or more of Pfs races 1, 2, 6, 7 and 9, and/or optionally to isolate UA0514 and/or other Pfs isolates, where representative seeds of said plant have been deposited under accession number NCIMB 42607.

[0211] In another embodiment, the invention contemplates use of a spinach plant comprising resistance to at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17) conferred by an introgression fragment obtainable from a seed deposited under accession number NCIMB 42607, or from progeny thereof (e.g. obtained by selfing), for generating a cultivated spinach plant comprising resistance to at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), optionally further to one or more of Pfs races 1, 2, 6, 7 and 9, and/or optionally to isolate UA0514 and/or other Pfs isolates.

[0212] Seeds

[0213] The invention provides a seed from which any plant of the invention can be grown. Furthermore, the invention provides a plurality of such seed. A seed of the invention can be distinguished from other seeds due to the presence of the RPF14 resistance gene, either phenotypically (based on plants having the RPF14 resistance phenotype) and/or using molecular methods.

[0214] In one aspect, a plurality of seed is packaged into a container (e.g. a bag, a carton, a can etc.). Containers may be any size. The seeds may be pelleted prior to packing (to form pills or pellets) and/or treated with various compounds, including seed coatings.

[0215] In an embodiment of the invention, the spinach seed is 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 for priming spinach seeds are well known in the art (see, e.g., Chen et al. 2010, Seed Sci. & Technol. 38: 45-57). In another embodiment, the spinach seed is treated with crop protection, or film coated, or pelleted. Film coating and treatment with crop protection are commonly combined, see e.g. US20170127670.

[0216] Plant Parts and Vegetative Reproductions

[0217] In a further aspect a plant part, obtained from (obtainable from) a plant of the invention is provided herein, and a container or a package comprising said plant part.

[0218] In a preferred embodiment the plant part is a leaf of a spinach plant of the invention or a plurality of leaves, or part of a leaf, preferably a harvested leaf. Such a leaf may be loose, bunched, fresh (e.g. in a container, for example a bag), frozen, blanched or boiled. Such a leaf may be fresh or processed, and they may be part of food or feed products. A leaf may be harvested in any stage of its development, preferred stages are baby leaf and mature leaf.

[0219] Other plant parts, of a plant of the invention, include a leaf, a part of a leaf, a stem, a part of a stem, a stalk, a part of a stalk, a shoot, a part of a shoot, a bud or a part of a bud, a cutting, a root, a part of a root, a root tip, a petiole, a part of a petiole, a cotyledon, a part of a cotyledon, a flower, a part of a flower, a petal, a part of a petal, a stamen, a part of a stamen, an anther, a part of an anther, pollen, a stigma, a part of a stigma, a style, a part of a style, an ovary, a part of an ovary, an ovule, a part of an ovule, a seed, a part of a seed, a seed coat, an embryo, a part of an embryo, a hypocotyl, an embryo sac, a fruit, a part of a fruit, a cell, a protoplast, callus, a microspore, meristem, cambium etc. The various stages of development of aforementioned plant parts are comprised in the invention.

[0220] Seeds include for example seeds produced on the plant of the invention after self-pollination 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.

[0221] In one aspect, the plant parts or seeds can be identified by the presence of the donor SNP nucleotide for the resistant donor nucleotide of SNP_01, which has Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0222] In a further aspect, the plant part is a plant cell. In still a further aspect, the plant part is a non-regenerable cell or a regenerable cell. In another aspect the plant cell is a somatic cell. In one aspect the cell is isolated from its natural location.

[0223] A non-regenerable cell is a cell which cannot be regenerated into a whole plant through in vitro culture. The non-regenerable cell may be in a plant or plant part (e.g. a leaf) of the invention. The non-regenerable cell may be a cell in a seed, or in the seed-coat of said seed. Mature plant organs, including a mature leaf, a mature stem or a mature root, comprise at least one non-regenerable cell. Maturing plant organs such as a baby leaf spinach leaf also comprise at least one non-regenerable cell.

[0224] Moreover, there is provided an 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 part described above, such as, for example and without limitation, a leaf, a part of a leaf, a stem, a part of a stem, a stalk, a part of a stalk, a shoot, a part of a shoot, a bud or a part of a bud, a cutting, a root, a part of a root, a root tip, a petiole, a part of a petiole, a cotyledon, a part of a cotyledon, a flower, a part of a flower, a petal, a part of a petal, a stamen, a part of a stamen, an anther, a part of an anther, pollen, a stigma, a part of a stigma, a style, a part of a style, an ovary, a part of an ovary, an ovule, a part of an ovule, a seed, a part of a seed, a seed coat, an embryo, a part of an embryo, a hypocotyl, an embryo sac, a fruit, a part of a fruit, a cell, a protoplast, callus, a microspore, meristem, cambium, a somatic cell, a non-reproductive cell or a reproductive cell.

[0225] Therefore, one aspect provides a cell culture or tissue culture comprising cells or tissues derived from a part a of a spinach plant of the species Spinacia oleracea comprising resistance to Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), wherein said resistance is conferred by a single gene introgressed from Spinacia turkestanica, which gene is linked to the resistant donor nucleotide of SNP_01, which comprises an Adenine (A) at nculeotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0226] In one aspect the cells or tissues can be identified by the presence of the donor genotype for SNP_01, which comprises Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0227] Also provided is a spinach plant regenerated from any of 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 RPF14 gene), i.e. retains the RPF14 gene (or the introgression fragment comprising the RPF14 gene) of the invention. This plant can also be referred to as a vegetative propagation of plants of the invention.

[0228] Also provided is a harvested leaf of a plant of the invention and a package comprising a plurality of leaves of one or more plants of the invention. These leaves thus comprise the RPF14 gene of the invention, detectable by e.g. linked molecular markers or phenotypically (for the originally used whole plant and/or regenerated plant). A leaf can be harvested at any developmental stage. Preferred developmental stages for harvesting a leaf are mature stage and baby-leaf stage.

[0229] 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 a leaf or a part of a leaf of a plant of the invention, or packaged frozen spinach.

[0230] A spinach plant of the invention or a progeny thereof retaining the Pfs resistance phenotype conferred by the RPF14 gene, and are optionally linked to the resistant donor nucleotide of SNP_01, which comprises Adenine (A) at nculeotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, and/or retaining the introgression fragment or sub-fragment comprising the RPF14gene and optionally said SNP_01 (or SEQ ID NO: 1), as present in NCIMB 42607, and a part of the aforementioned plant, can be suitably packed for 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: a leaf, a cutting, pollen, a part of a leaf, and the like.

[0231] 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. Encompassed are therefore also 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. For example, 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.

[0232] Plants and Progeny

[0233] In another embodiment, 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 regenerated or vegetatively propagated) plant comprises resistance to at least Pfs races 8 and 10 to 16, preferably against at least 8 and 10 to 17 (optionally further to one or more of Pfs races 1, 2, 6, 7, and 9, and/or optionally against isolate UA0514 and/or other Pfs isolates (as conferred by the RPF14gene, optionally linked to the resistant donor nucleotide of SNP_01, which comprises Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0234] In one aspect, a progeny plant of a spinach plant of the invention is a progeny plant that retains the RPF14 resistance gene linked to the resistant donor nucleotide of SNP_01, which has an Adenine (A) at nculeotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0235] In another aspect, the progeny plant is a spinach plant of the species Spinacia oleracea comprising resistance to Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), wherein said resistance is conferred by a single gene RPF14 introgressed from Spinacia turkestanica, which gene is in one aspect linked to the resistant donor nucleotide of SNP_01, which is Adenine (A) at nucleotide 114 of SEQ ID NO: 1 or an Adenine at the equivalent position of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Preferably, presence of RPF14can be identified by aforementioned donor nucleotide for SNP_01 and/or another marker linked to RPF14 or to the introgression fragment.

[0236] As mentioned before, whether or not a plant, progeny or vegetative propagation comprises the Pfs resistance phenotype as conferred by the RPF14gene 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 introgression), chromosome painting, etc.

[0237] Furthermore, the invention provides for progeny comprising or retaining the Pfs resistance phenotype (conferred by the RPF14 gene), such as progeny obtained by, e.g., selfing 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. In particular, the invention provides for progeny that retain the RPF14 gene (conferring the Pfs resistance phenotype) of (as found in) NCIMB 42607. In one aspect the invention provides for a progeny plant comprising the RPF14 resistance, such as a progeny plant that is produced from a spinach plant comprising the RPF14 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. In one embodiment, 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. Similarly, 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.) 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 RPF14gene and the Pfs resistance phenotype conferred by it and has the desired trait.

[0238] In one aspect haploid plants and/or double haploid plants of plant of the invention are encompassed herein, which comprise resistance to at least Pfs races 8 and 10 to 16 (preferably against at least 8 and 10 to 17), optionally further to one or more of Pfs races 1, 2, 6, 7, and 9, and/or optionally against isolate UA0514 and/or other Pfs isolates, as conferred by the RPF14 gene or by the introgression fragment comprising the RPF14 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.

[0239] In another embodiment the invention 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. In a further embodiment the invention relates to seed produced according to this method and/or a spinach plant produced by growing such seed. Thus, 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 resistance to at least Pfs 8 and 10 to 16 (preferably against at least 8 and 10 to 17), optionally further to one or more of Pfs races 1, 2, 6, 7, and 9, and/or optionally against isolate UA0514 and/or other Pfs isolates, due to the presence of the RPF14gene.

[0240] Thus, in one aspect progeny of a spinach plant of the invention are provided, wherein the progeny plant is produced by selfing, crossing, mutation, double haploid production or transformation and wherein the progeny retain the RPF14 resistance gene (and phenotype conferred by it) described herein, e.g. obtainable by crossing a spinach plant, grown from seeds deposited under accession number NCIMB 42607, with another spinach plant. In other words, in one aspect 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 42607 is retained in the progeny plants.

[0241] Molecular markers may also be used to aid in the identification of the plants (or plant parts or nucleic acids obtained therefrom) comprising the RPF14 resistance gene or locus. For example, one can develop one or more molecular markers which are closely genetically physically linked to the RPF14 resistance gene or locus. This can be done by crossing a resistant spinach plant (comprising RPF14) with a susceptible spinach plant and developing a segregating population (e.g. F2 or backcross population) from that cross. 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 RPF14 resistance gene or locus can be identified. By BLAST analysis against SpinachBase the physical location on chromosome 3 can be determined. If flanking markers are identified (either side of the RPF14 gene), the physical region of chromosome 3 where RPF14 is located between the markers can be identified.

[0242] Molecular markers which are closely linked to RPF14 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 RPF14 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). Preferably, linked markers are used in MAS. One sequence that can be used as a marker is the sequence comprising SNP_01 as described. More preferably, flanking markers are used in MAS, i.e. one marker on either side of the RPF14 gene or locus.

[0243] Using SEQ ID NO: 1 and the deposited seeds disclosed herein, the skilled person can also identify the sequence of the RPF14 gene itself using methods known in the art. For example, sequencing the chromosome 3 region and comparing the sequence to the sequence in e.g. SpinachBase, can be used to identify open reading frames on the introgression fragment, in order to identify the RPF14 gene itself. Modification of the RPF14 gene, e.g. by CRISPR-Cas9, can be used to prove the function of the gene. The skilled person can thus also generate plants comprising induced mutations in the RPF14 gene (e.g. in the promoter, protein coding sequence, other regulatory sequences). Plants comprising induced mutations in the RPF14 gene are encompassed herein.

[0244] In one aspect a method for screening, and optionally selecting, spinach seeds, plants or plant parts or DNA from such seeds, plants or plant parts for the presence of one or more markers linked to the RPF14 gene is provided.

[0245] In other aspects, the RPF14 gene is detectable using one or more nucleic acid probes, nucleic acid primers, or a combination thereof.

[0246] Thus, in one aspect the RPF14 gene is detectable by one or more nucleic acid probes, which hybridize to genomic DNA obtained from a plant or plant part comprising the RPF14 gene using stringent hybridization conditions.

[0247] A nucleic acid probe may for example be a DNA molecule which comprises SEQ ID NO: 1 (or which comprises Adenine at the location equivalent to 114 of a sequence comprising at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1) or its complement sequence. In another aspect the RPF14 gene is detectable by one or more nucleic acid primers, which amplify genomic DNA linked to the RPF14 gene. For example, the primers may amplify a nucleic acid molecule comprising aforementioned SEQ ID NO: 1, or a sequence comprising at 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 having Adenine at the position equivalent to114. Suitable primers are for example the 70 to 100 bp upstream and 70 to 100 bp downstream of the SNP marker can be selected to design a forward and a reverse primer, which amplify the marker. The primers can be used e.g. for SNP genotyping, e.g. in a KASP-assay for detecting the SNP genotype for SNP_01.

[0248] 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 RPF14 gene or locus) in a plant or plant part can also be useful for breeding purposes.

[0249] Deposit Information

[0250] A representative number of Spinacia oleracea NCIMB 42607 were deposited by Nunhems B.V. under the Budapest Treaty on 12 Jul. 2016, at the NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom (NCIMB). Access to the deposit will be available during the pendency of this application to persons determined by the Director of the

[0251] U.S. Patent Office to be entitled thereto upon request. Subject to 37 C.F.R. 1.808(b), all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent. The deposit will be maintained for a period of 30 years, or 5 years after the most recent request or for the enforceable life of the patent whichever is longer, and will be replaced if it ever becomes nonviable during that period. Applicant does not waive any rights granted under this patent on this application or under the Plant Variety Protection Act (7 USC 2321 et seq.).

[0252] Various modifications and variations of the described products and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in plant breeding, chemistry, biology, plant pathology or related fields are intended to be within the scope of the following claims.

[0253] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

EXAMPLES

[0254] 1Selection of a Wild Donor and Crossing the RPF14 Gene from the Wild Donor into Cultivated Spinach

[0255] Several wild accessions were tested for resistance to infection with Peronospora farinosa f. sp. spinaciae races 1 to 16 and isolate UA0514. A Spinacia turkestanica accession was found to be resistant to at least Pfs races 8 and 10 to 16 and selected.

[0256] The selected plant (resistance donor) was crossed with a cultivated spinach plant, that did not have any known background resistance to Pfs. Progeny plants were tested for Pfs resistance to at least Pfs races 8 and 10 to 16 as exhibited by the selected plant (donor), and the resistant progeny plants were selected and backcrossed with said cultivated spinach plants and selfed for several generations to generate a line comprising the introgression fragment in homozygous form. Thus, the resistance from the Spinacia turkestanica donor was introgressed into cultivated spinach plants. Seeds of a plant line thus obtained were deposited with the NCIMB under number NCIMB 42607.

[0257] 2Pfs Resistance Phenotype of Cultivated Spinach Comprising the RPF14 Gene

[0258] The resistance to Peronospora farinosa infection was tested with the help of a differential set (obtainable from a.o. Naktuinbouw, The Netherlands).

[0259] Spinach plants grown from NCIMB 42607 seed (comprising the RPF14 gene) were planted along with the differential set and spinach plants from other genotypes serving as checks in trays containing BVB substrate (Euroveen, Grubbenvorst), and covered with Agra-vermiculite (Pull,

[0260] Rhenen). Per test at least 10 plants from one genotype each where tested in one or two replications. The trays were placed in a climate cell at 12 C./15 C. (day/night) with a 12 h photoperiod. Plants were inoculated by spraying a sporangial suspension (2.510.sup.5/ml) of a pathogenic race of Peronospora farinosa f. sp. spinaciae 14 days after seeding. In this manner pathogenic races were assayed. 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%.

[0261] 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). A plant line exhibiting evidence of sporulation was considered susceptible. A plant line where at least 85% of individuals of the line did not exhibit sporulation was 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. Any genotype with <15% of plants being categorized as susceptible (i.e. with more than 85% of plants not showing sporulation) were considered as a resistant genotype.

[0262] The new resistance gene, RPF14, as present in NCIMB 42607 in homozygous form, was found to confer resistance to Pfs 1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 and isolate UA0514. Table 2 shows the resistances of spinach plants grown from NCIMB 42607 seed (comprising RPF14 homozygously) against Pfs race 1-16 and isolate UA0514.

[0263] Lines homozygous for RPF11, RPF12 or RPF13 were found not to be resistant to Pfs race 16.

TABLE-US-00003 Line comprising gene Pfs 8 Pfs 9 Pfs 10 Pfs 11 Pfs 12 Pfs 13 Pfs 14 Pfs 15 Pfs 16 RPF11 (homozygous) + RPF12 (homozygous) + RPF13 (homozygous) +

[0264] The susceptible and resistant differential varieties of Table 1 were used as checks and behaved as expected according to the standard as given in Table 1 (data not shown).

TABLE-US-00004 TABLE 2 Disease resistance of RPF14 Line Pfs 1 Pfs 2 Pfs 3 Pfs 4 Pfs 5 Pfs 6 Pfs 7 Pfs 8 Pfs 9 Pfs 10 Pfs 11 Pfs 12 Pfs 13 Pfs 14 Pfs 15 Pfs 16 Pfs 17 UA0514 NCIMB 42607 + + + (comprising RPF74 homozygous) Legend: ( means resistant; + means susceptible;)

[0265] 3Introgression of the RPF14 Resistance Trait into Other Spinach Plants

[0266] In another experiment, a spinach plant grown from NCIMB 42607 seed was crossed (as a father) with a different spinach plant susceptible to the race to be tested. Plants of the F1 population were tested for Pfs resistance against Pfs races 8 and 10 to 16, as described in Example 2. It was observed that the heterozygous F1 plants retained the resistance against Pfs races 8 and 10 to 16, and thus it was concluded that the resistance to these races is dominant. In the F2 population the resistance to these races segregates in a 3 (resistant): 1 (susceptible) ratio.

[0267] 4Development of a Marker Linked to RPF14 and Alignments

[0268] An F2 population was developed by crossing spinach plant of NCIMB 42607 (comprising the RPF 14 gene) with a spinach plant that does not have the RPF14 resistance gene and does not have background resistance to Pfs. Linkage mapping was conducted, and a Single Nucleotide Polymorphism marker (SNP), SNP_01 shown in Table 3, was identified linked to the RPF14 gene.

TABLE-US-00005 TABLE3 ASNPmarkerlinkedtotheRPF14gene SNPand SNPgenotypein SNPgenotype SequencecomprisingSNP_01 nucleotide spinachplant ofthe atnucleotide114 position comprisingthe recurrent (nt)inthe donor parent(no sequence introgression introgression fragmentin fragment) homozygous form SNP_01at AA GG GCTCGTATHACTTAGGGCTHGAGCHYGAAYCT nucleotide CCATCACCAACAAGGGCAGGCAACGATCTCG 114ofSEQ AAAGATTATTCAAATYGTAAAAHGAAGCATAA IDNO:1 TTCGCATTATTATTATTC[A/G]ACATTCTCAATY TATCATAATTAGACSGGCGTATGCCACCATAC CCGTGTYTAAACCCATGGATCGACCATTGATA ACCGGGTTTATGAATCATTGAAT (SEQIDNO:1and2)

[0269] SEQ ID NO: 1 comprises Adenine at nucleotide 114 and SEQ ID NO: 2 comprises a Guanine at nucleotide 114. As mentioned before, the recurrent parent is susceptible.

[0270] Other spinach germplasm was screened for the genotype at SNP_01 and another susceptible line was found, which comprises the susceptible nucleotide for SNP_01, but varies in that it also comprises six additional nucleotides upstream (5) of SNP_01 (nucleotides GTTATT). That sequence is included as SEQ ID NO: 3 and is 219 instead of 213 nucleotides long. Due to these six extra nucleotides, SNP_01 is not located at nucleotide 114 in SEQ ID NO: 3, but at nucleotide 120, which is the equivalent position of SNP_01 in SEQ ID NO: 3 as seen from the pairwise alignment (see FIG. 1; SNP_01 in bold).

[0271] SEQ ID NO: 1 and 2 were also used in a BLAST analysis against SpinachBase genome sequence (of Chinese spinach line SP75). This analysis showed that SEQ ID NO: 1 and 2 are located on chromosome 3, starting at nucleotide 607896 and ending at nucleotide 607679, with the SNP_01 being at nucleotide 607778 (comprising a G at this nucleotide) in SP75.

[0272] In a pairwise alignment of the genome sequence of SpinachBase with SEQ ID NO: 1, the two sequences have 90% sequence identity (pairwise alignment using Emboss program Needle, default parameters) (see FIG. 2, SNP_01 in bold). It is also evident that the genomic sequence in SpinachBase does comprise the six extra nucleotides found in the susceptible spinach line in SEQ ID NO: 3. A pairwise alignment of SEQ ID NO: 3 with the genomic sequence in SpinachBase showed a sequence identity of 93.2%. This indicates that there is variability in the S. o/eracea genome.

[0273] FIGS. 1 and 2 show that the Adenine at nucleotide 114 of SEQ ID NO: 1 (present on the introgression fragment from S. turkestanica) can be identified in sequences which are not 100% identical to SEQ ID NO: 1, by pairwise sequence alignment of SEQ ID NO: 1 with other sequences, e.g. sequences comprising at least 90% sequence identity to SEQ ID NO: 1. The nucleotide position is such sequences is referred herein to as the nucleotide position equivalent to position 114 in SEQ ID NO: 1.

[0274] The Adenine at nucleotide 114 in SEQ ID NO: 1 (SNP_01), or at the equivalent nucleotide in a sequence comprising at least 90% sequence identity to SEQ ID NO: 1 can be used to select plants and plant parts comprising the introgression fragment on which RPF14 is located.

[0275] 5Tests for Markers for Other Resistance Genes

[0276] WO2015054339 is a patent application, describing an introgression from S. tetrandra which comprises a Quantitative Trait Locus (QTL) conferring resistance to at least Pfs races 7, 10, 11, 12, 13, and 14. The application also discloses S. tetrandra flanking sequences, SEQ ID NO:1 and SEQ ID NO:2 which flank the QTL in the S. tetrandra donor described in WO2015054339, i.e. they flank said S. tetrandra gene conferring resistance to at least Pfs races 7, 10, 11, 12, 13, and 14. SEQ ID NO:1 of WO2015054339, hereafter referred to as left S. tetrandra flanking marker, was added to this application as SEQ ID NO:4, and SEQ ID NO:2 of WO2015054339, hereafter referred to as right S. tetrandra flanking marker, was added to this application as SEQ ID NO:5.

[0277] The plant line deposited by the instant inventors was tested for the presence of said left and right S. tetrandra flanking markers. For each of the two flanking sequence regions described in the patent two primer pairs were designed, amplifying the corresponding region in DNA of the spinach plant grown from NCIMB 42607. In total 8 PCR primers were ordered and checked In silicio using JCeasar for resulting into a PCR fragment within the region.

[0278] PCR was performed on DNA of the spinach plant grown from NCIMB 42607 seed, using the primer pair combination for the left S. tetrandra flanking marker. The resulting PCR products were verified on agarose gel for the expected fragment length. The fragments had the expected size and were sequenced. The sequenced fragments of the material were aligned into contigs for each of the two flanking sequence regions in Sequencher. Based on these contigs, the spinach plant grown from NCIMB 42607 seed does not comprise the left S. tetrandra flanking marker. Instead, the NCIMB 42607 seed and plants grown from it were found to have another sequence (S. oleracea sequence), added to this application as SEQ ID NO:6. The alignment between the two sequences is shown in FIG. 3. It is clear that the left S. tetrandra flanking sequence is not present in the seeds deposited.

[0279] A second PCR was performed on DNA of the spinach plant grown from NCIMB 42607 seed, using the primer pair combination for the right S. tetrandra flanking marker. The resulting PCR products were verified on agarose gel for the expected fragment length. The fragments had the expected size and were sequenced. The sequenced fragments of the material were aligned into contigs for each of the two flanking sequence regions in Sequencher. Based on these contigs, the spinach plant grown from NCIMB 42607 seed does not have the right S. tetrandra flanking marker. Instead, NCIMB 42607 seed and plants grown from it were again found to have another sequence, added to this application as SEQ ID NO:7 (S. oleracea). The alignment between the two sequences is shown in FIG. 4. It is clear that the right S. tetrandra flanking sequence is not present in the seeds deposited. Please show me what you aligned and how

[0280] Thus, neither the left nor the right S. tetrandra flanking marker is present in NCIMB 42607. NCIMB 42607 comprises the S. oleracea sequences SEQ ID NO: 6 and 7 in the region of the chromosome. Obviously, other cultivated spinach lines or varieties according to the invention may comprise SEQ ID NO: 6 and/or SEQ ID NO: 7 in their genome, or a sequence comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to either of SEQ ID NO: 6 or 7.

[0281] Interestingly, when doing a BLAST analysis in SpinachBase using these sequences, they appear to located on chromosome 3, and not on chromosome 6 as mentioned in the patent application.

TABLE-US-00006 SpinachBase chromosome 3-first and last nucleotide of the BLAST alignment SEQ ID NO: 4 1418829-148725 (S. tetrandra patent) SEQ ID NO: 6 1418964-1418666 (100% identical) (S. oleracea in NCIMB42607) SEQ ID NO: 5 711828-711679 (S. tetrandra) SEQ ID NO: 7 711906 -711670 (100% identical) (S. oleracea in NCIMB42607)

[0282] The S. tetrandra QTL therefore appears to lie between 0.7 Mb and 1.41 Mb of chromosome 3.

[0283] 6Fine Mapping of RPF14

[0284] A further segregating population will be developed by crossing spinach plant grown from NCIMB 42607 seed (comprising the RPF14 gene) with a spinach plant that does not comprise the RPF14 resistance gene and does not comprise background resistance to Pfs. Also further SNPs will be added to the chromosome region where RPF14 is found. Further mapping will be conducted, generating more Single Nucleotide Polymorphism marker (SNP) markers linked to the RPF14 gene.

REFERENCES

[0285] Correll et al. 2011, Eur J Plant Pathol 129: 193-205

[0286] Correll et al. 2010, Guidelines for Spinach Downy Mildew: Peronspora ferinosa f.sp. spinaciae (Pfs) found on the website of the ISF

[0287] Smith, P. G. and M. B. Zahara. 1956. New spinach immune to mildew. Calif. Agr. 10:15.

[0288] Smith, P. G., R. E. Webb, and C. H. Luhn. 1962. Immunity to race 2 of spinach downy mildew. Phytopathology 52:597-599.

[0289] Smith, P. G., R. E. Webb, A. M. Millett, and C. H. Luhn. 1961. Downy mildew on spinach. Calif. Agr. 15:5.

[0290] Brandenberger et al. (1992) HORTSCIENCE 27(20):1118-1119.

[0291] Plantum press release, Denomination of Pfs: 16, a new race of downy mildew in spinach Mar. 15 2016

[0292] International Seed Federation Guidelines for Spinach Downy Mildew Peronospora farinosa f. sp. spinaciae (Pfs) Jim Correll, Lindsey du Toit, Steven Koike, and Kees van Ettekoven, dec 2015; http://www.worldseed.org/isf/differential_hosts.html

[0293] Xu, C. et al. (2017, Nat. Commun. 8,15275 doi: 10.1038/ncomms15275) Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions (2017)