Novel pumpkin plant resistant to Papa Earring Spot Virus (PRSV)

Abstract

This invention relates to a pumpkin (Cucurbita Pepo L.) plant that exhibits high resistance to papaya ring spot virus infection, obtained by gene transfer from C. ecuadorensis, located on chromosome 10. The invention also relates to the seeds and parts of the plant, such as fruits. Furthermore, the invention relates to methods for producing and using such seeds and plants. The invention also relates to a novel gene sequence associated with the high resistance, and a molecular marker associated with the novel gene sequence. The invention further relates to markers and their use in marker-assisted breeding.

Description

Technical Field

[0001] The present invention relates to a novel pumpkin plant body that exhibits a high increase in resistance to papaya ringspot virus infection. The present invention also relates to seeds and parts of said plant body, such as fruits. The present invention further relates to methods of producing and using such seeds and plants. The present invention also relates to novel gene sequences associated with said high resistance, and molecular markers associated with said novel gene sequences. The present invention further relates to markers and their use in marker-assisted breeding.

Background Art

[0002] Pumpkin (Cucurbita pepo L.) is an important specialty crop native to North America. The genus Cucurbita, which is cultivated and used for food, is a common crop in many major agricultural production regions, and the world production in 2019 was reported to be 22,900,826 tons (based on data provided by the Food and Agriculture Organization). In 2019, only the production in the United States was equivalent to a maximum of 220 million US dollars (USDA Vegetables 2019 Summary).

[0003] Plant pathogens are known to cause significant damage to important crops including pumpkins, resulting in significant agricultural losses that widely affect both food supply and other industries that depend on plant materials. Therefore, there has long been a need to reduce the occurrence and / or impact of agricultural pests on crop production. This applies to several viruses such as cucumovirus (cucumber mosaic virus), begomovirus (pumpkin leaf curl virus), and potyviruses such as zucchini yellow mosaic virus, watermelon mosaic virus, and papaya ringspot virus.

[0004] The Potyvirus group (named after its prototype member, potato Y virus PVY) is the largest of the 34 currently recognized groups and families of plant viruses (Ward & Shukla, 1991). This group includes at least 180 confirmed and potential members (30% of all known plant viruses) that cause significant losses to crops, pasture crops, horticultural crops, and ornamental crops (Ward & Shukla, 1991). Papa Earling Spot Virus (PRSV; Potyviridae, Potyvirus) is one of the most harmful Potyviruses and is known to affect various Cucurbitaceae crops, including pumpkins (Cucurbita pepo).

[0005] In markets across Europe, Africa, the Middle East, and North America, various types of commercially available summer pumpkin (C. pepo) are expected to exhibit moderate resistance to PRSV. Some level of resistance has been found in Cucurbita species such as Cucurbita ecuadorensis, Cucurbita maxima, Cucurbita foetidissima, and Cucurbita moschata (Provvidenti et al., 1978; Maluf et al., 1986), and several commercially available varieties of Cucurbita pepo are said to have moderate resistance to PRSV. Nevertheless, because viruses tend to overcome existing resistance factors, and the incidence of pottyvirus infections is increasing in resistant or moderately resistant varieties, stable resistance to pottyvirus is a crucial driving force in pumpkin breeding. For these reasons, using gene pyramidalization to target specific pathogens and enhance resistance to groups of pottyviruses is one strategy. This invention addresses this need by providing novel alleles related to PRSV resistance. [Overview of the project] [Means for solving the problem]

[0006] The present invention relates to a novel cultivated Cucurbita pepo plant comprising gene transfer of a QTL associated with increased resistance to papaya ringing spot virus (PRSV) and the corresponding basal QTL allele into an elite pumpkin plant. The PRSV-resistant QTL and its basal allele sequence (QTL10), located on chromosome 10, are semi-dominant, and therefore one copy of this allele is sufficient to provide an improved PRSV-resistant phenotype.

[0007] Overall, the characteristics of the pumpkin plants with improved PRSV resistance disclosed in this invention provide pumpkin growers with a novel solution that enhances economic and commercial efficiency when developing pumpkin varieties in fields under PRSV pressure.

[0008] In the first embodiment, the present invention relates to a cultivated pumpkin plant resistant to papaya earring spot virus (PRSV) infection, preferably a cultivated Cucurbita pepo plant, wherein its genome contains a gene transfer sequence derived from Cucurbita ecuadorensis that confers resistance to PRSV, the gene transfer sequence being located on chromosome 10 and containing the following SNP markers: a) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; b) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of SEQ ID NO: 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; g) A C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) A C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for SNP marker 11 at position 36 of SEQ ID NO: 11; and / or l) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12. The present invention provides a plant body containing at least one of the following.

[0009] In a further embodiment, the gene transfer sequence includes at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and / or SEQ ID NO: 12, or a sequence that is at least 80% identical to one or more of the aforementioned sequences.

[0010] In a further embodiment, the plant is homozygous for the at least one SNP marker.

[0011] In a further embodiment of the present invention, the plant of any of the above embodiments further comprises a Zn allele located on chromosome 16 that is associated with zucchini yellow mosaic virus (ZYMV) resistance, wherein the Zn allele comprises a G genotype that is homozygous for SNP marker 13 at position 119 of SEQ ID NO: 13.

[0012] In further embodiments, the gene transfer sequence is contained in the Cucurbita pepo plant 22SQE800260 (whose representative seeds are deposited under NCIMB accession number 44148), or its offspring or ancestors.

[0013] In a further embodiment, the present invention provides a plant according to any of the above embodiments, which is obtained by crossing a Cucurbita pepo plant 22SQE800260, whose representative seeds are deposited under NCIMB accession number 44148, or its offspring or ancestor, with a pumpkin plant that does not contain the gene transfer sequence that confers PRSV resistance.

[0014] In a further embodiment, the present invention provides a plant according to any of the above embodiments, wherein the plant is an inbred, diploid haploid, diploid, or hybrid plant.

[0015] A further embodiment of the present invention is to provide a plant portion of a plant body according to any one of the embodiments described above.

[0016] In further embodiments, the present invention provides seeds that produce a plant body or plant part according to any one of the embodiments described above.

[0017] In further embodiments, the present invention relates to a method for producing cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, that are resistant to PRSV infection. a) A step of crossing a plant according to any one of claims 1 to 7 with a cultivated pumpkin plant lacking the gene transfer sequence that confers PRSV resistance, b) A step of selecting a progeny plant containing the gene transfer sequence located on chromosome 10 that confers resistance to PRSV, the following SNP marker: i) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; ii) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of sequence number 2; iii) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; (iv) a G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of SEQ ID NO: 4; (v) a G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of SEQ ID NO: 5; (vi) a G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of SEQ ID NO: 6; (vii) a C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of SEQ ID NO: 7; (viii) an A genotype that is heterozygous or homozygous for SNP marker 8 at the position corresponding to position 36 of SEQ ID NO: 8; (ix) a C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of SEQ ID NO: 9; (x) a G genotype that is heterozygous or homozygous for SNP marker # 10 at the position corresponding to position 36 of SEQ ID NO: 10; (xi) an A genotype that is heterozygous or homozygous for SNP marker 11 at the position corresponding to position 36 of SEQ ID NO: 11; and / or (xii) an A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of SEQ ID NO: 12 comprising a selection step of detecting at least one of and thereby providing a method for generating a plant body with enhanced resistance to PRSV.

[0018] In a further embodiment, the present invention is a method according to the foregoing embodiment, (c) self - propagating the selected progeny or crossing the selected progeny with another pumpkin plant body to generate further progeny further comprising a method.

[0019] In a further embodiment, the present invention provides a method according to the method of the foregoing embodiment, further comprising selecting additional progeny and selfing / crossing for an additional 2 to 10 generations.

[0020] In a further embodiment, the present invention provides a method according to the method of the foregoing embodiment, further comprising detecting a G genotype that is heterozygous or homozygous for SNP marker 13 at a position corresponding to position 119 of SEQ ID NO: 13.

[0021] In a further embodiment, the present invention provides a method according to the method of the foregoing embodiment, wherein the plant body in step a) is a Cucurbita pepo plant body 22SQE800260 or its progeny or ancestor whose representative seeds are deposited under NCIMB accession number 44148.

[0022] In a further embodiment, the present invention provides a method for generating an F1 pumpkin plant body resistant to PRSV, comprising crossing an inbred pumpkin plant body, which is a plant body according to any one of the foregoing embodiments, with a different inbred pumpkin plant body to generate F1 hybrid progeny.

[0023] In a further embodiment, the present invention provides a method for identifying a cultivated pumpkin plant body, preferably a cultivated Cucurbita pepo plant body, that is resistant to PRSV infection and has at least one copy of a gene transfer sequence conferring the PRSV resistance, the following SNP markers: a) An A genotype that is heterozygous or homozygous for SNP marker 1 at a position corresponding to position 36 of SEQ ID NO: 1; b) A G genotype that is heterozygous or homozygous for SNP marker 2 at a position corresponding to position 36 of SEQ ID NO: 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at a position corresponding to position 36 of SEQ ID NO: 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; g) A C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) A C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for SNP marker 11 at position 36 of SEQ ID NO: 11; and / or l) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12. The present invention provides a method for identifying pumpkin plants that exhibit resistance to PRSV, comprising the step of detecting at least one of the following.

[0024] In further embodiments, the present invention provides a method according to the above-described embodiments, further comprising selecting a pumpkin plant containing one or more SNP markers, and crossing the selected pumpkin plant with a second pumpkin plant to produce offspring pumpkin plants containing at least one of the SNP markers and exhibiting resistance to PRSV.

[0025] In further embodiments, the present invention provides a method for producing pumpkin seeds, comprising growing a pumpkin plant from the seeds of the above embodiments and causing the plant to produce further pumpkin seeds.

[0026] In further embodiments, the present invention relates to a method for evaluating the genotype of cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, that exhibit resistance to PRSV, a) A step of preparing a sample from the plant body, b) A step of detecting a QTL locus located on chromosome 10 and associated with PRSV resistance in the sample, wherein the QTL locus is identified by the following SNP marker: i) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; ii) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of sequence number 2; iii) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; iv) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; v) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; vi) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; vii) A C genotype that is heterozygous or homozygous for SNP marker 7 at position 36 of sequence number 7; viii) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; ix) A C genotype that is heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; x) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; xi) A genotype that is heterozygous or homozygous for SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or xii) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12; and / or xiii) Any other DNA marker associated with the QTL locus, flanked by SNP markers 1 and 12 A step including at least one of the following This provides a method that includes this.

[0027] In further embodiments, the present invention provides a method for identifying gene transfer sequences associated with high resistance to PRSV in cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, comprising the step of detecting an allele of at least one DNA marker genetically associated with the QTL locus associated with the high resistance to PRSV in the plant, wherein the allele is located within 10 cM, preferably within 5 cM, of the QTL locus located on chromosome 10 in a genomic region flanked by SNP markers 1 and 12.

[0028] In further embodiments, the present invention relates to the method according to the above-described embodiments, wherein the QTL locus is the following SNP marker a) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; b) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of SEQ ID NO: 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; g) A C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) A C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for SNP marker 11 at position 36 of SEQ ID NO: 11; and / or l) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12. The present invention provides a method that can be identified by at least one of the following.

[0029] In further embodiments, the present invention provides a method according to the above embodiments, further comprising the step of selecting a cultivated pumpkin plant, preferably a cultivated Cucurbita pepo plant, containing the gene transfer sequence.

[0030] In a further embodiment, the present invention provides a method for identifying cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, that exhibit high resistance to PRSV by identifying QTLs associated with high resistance to PRSV, a) A step of detecting at least one DNA marker derived from a pumpkin plant (this DNA marker is associated with a chromosomal segment associated with high resistance to PRSV), wherein the chromosomal segment is flanked on both sides by SNP markers having at least 80% sequence identity to SEQ ID NOs: 1 and 12; b) The step of identifying the pumpkin plant containing the at least one DNA marker. This provides a method that includes [something].

[0031] In further embodiments, the present invention relates to a method for identifying a wild pumpkin source with a PRSV-resistant trait on chromosome 10, a) To provide one or more wild pumpkin varieties; b) Screening one or more wild pumpkin strains using a kit that detects at least one of the SNP markers listed in Table 5, c) Identify a wild pumpkin strain containing at least one of the SNP markers selected from the list in Table 5. This provides a method that includes this.

[0032] In a further embodiment, the present invention provides the use of any of Sequence IDs 1 to 12 for screening a population of pumpkin plants for the presence of a QTL locus located on chromosome 10 that is associated with high PRSV resistance. [Brief explanation of the drawing]

[0033] [Figure 1] The following are representative PRSV pathology images from the disease scale used and described in Example 2C, which uses a 1-9 evaluation scale ranging from susceptibility to resistance. [Figure 2] The results of PRSV virus pathology tests performed during the seedling stage, in which cotyledons exhibiting symptoms of susceptibility checks (a) and resistant plants possessing the described invention (b) were mechanically inoculated, are shown. [Figure 3]This plot shows the resistance levels of PRSV virus-converted strains and heterozygous material sets, grouped based on the status of PRSV-resistant QTLs and ZYMV-Zn-resistant QTLs, compared to susceptibility checks (P1 = homozygous resistance, H1 = heterozygous, N = homozygous susceptibility). [Modes for carrying out the invention]

[0034] definition Technical terms and expressions used within the scope of this application should generally be given the meanings that apply to the relevant art of plant breeding and cultivation, unless otherwise indicated below herein.

[0035] As used herein and in the appended claims, the singular forms “a,” “an,” and “it” include multiple referents unless otherwise specified in the context. For example, a reference to “plant body” includes one or more plant bodies, and a reference to “cell” includes mixtures such as cells and tissues.

[0036] Within the scope of this invention, "cultivated pumpkin" or "elite pumpkin" plants are understood to refer to plants that are no longer in their natural state but have been developed and domesticated through human care for agricultural use and / or human consumption, excluding wild pumpkin strains such as C. ecuadorensis strains. As an example, in embodiments, the cultivated pumpkin plants or elite pumpkin plants according to this invention can produce delicious edible fruit. Alternatively or in addition, the cultivated pumpkin plants are hybrid plants. Alternatively or in addition, the cultivated pumpkin plants may be C. pepo subsp. pepo var. cylindrica, C. pepo subsp. pepo var. ionga, C. pepo subsp. pepo var. pepo, C. pepo subsp. Ovifera var. recticollis, C. pepo subsp. Ovifera var. torticollia, or C. pepo subsp. Ovifera var. clypeata, preferably C. pepo subsp. pepo var. cylindrica (var. cylindrica) plant, more preferably cultivated zucchini pumpkin plant.

[0037] Within the scope of this invention, "allele" is understood to mean different forms of a gene or any kind of identifiable genetic determinant, e.g., alternative or variant forms of various genetic units identical to or associated with a QTL (these are choices in heredity because they are located at the same locus on homologous chromosomes). Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations, or deletions, or of genetic regulation caused by, for example, chemical or structural modifications, transcriptional regulation, or post-translational modification / regulation. In diploid cells or organisms, two alleles of a given gene or genetic element typically occupy corresponding loci on a pair of homologous chromosomes.

[0038] Relatively speaking, the terms “improved PRSV resistance” or “high PRSV resistance” are understood herein to mean that a plant according to the present invention, for example, that includes a gene transfer sequence derived from a Cucurbita ecuadorensis plant that confers resistance to PRSV, wherein the gene transfer sequence is located on chromosome 10 and includes at least one of SNP markers 1 to 12, has greater tolerance or resistance to PRSV varieties compared to a plant lacking the gene transfer sequence.

[0039] Within the scope of the present invention, "improved PRSV resistance" is understood to mean pumpkin plants that exhibit statistically significant improved resistance to PRSV compared to control pumpkin plants lacking the gene transfer sequence of the present invention (e.g., those described in the Examples section), with P<0.05 or P<0.01 using standard errors and / or Student's tests.

[0040] Within the scope of this invention, "phenotype" is understood to refer to a distinguishable characteristic of a genetically controlled trait.

[0041] Within the scope of this invention, "control pumpkin plant" is understood to mean a pumpkin plant having the same genetic background as the cultivated pumpkin plant of this invention, where the control plant does not have the gene transfer sequence of this invention related to improved PRSV resistance. In particular, the control pumpkin plant is a pumpkin plant belonging to the same plant variety but not containing the gene transfer sequence of this invention. The control pumpkin plant is grown for the same period and under the same conditions as the cultivated pumpkin plant of this invention. In this specification, plant variety is understood according to the definition of UPOV. Therefore, the control pumpkin plant may be a near-isogenic line, an inbred line, or a hybrid, but having the same genetic background as the pumpkin plant of this invention, except that the control plant does not contain the gene transfer sequence of this invention related to improved PRSV resistance.

[0042] The term "trait" refers to a characteristic or phenotype. In relation to the present invention, the PRSV-resistant trait is an improved PRSV-resistant trait. Traits can be dominant or recessive, or inherited partially, semi-dominant, or incompletely dominant. In relation to the present invention, the PRSV-resistance-constituting sequence, which is transferred from C. ecuadorensis and located on chromosome 10, is semi-dominant. Pumpkin plants of the present invention can therefore be homozygous or heterozygous with respect to the trait. Furthermore, the trait can be monogenic or polygenic, or may result from the interaction of one or more genes with the environment. In relation to the present invention, the PRSV-resistance-constituting gene transfer sequence located on chromosome 10 is sufficient on its own to confer an improved PRSV-resistant trait. Combination with additional potivirus resistance genes against other potiviruses further confers a higher level of resistance and avoids the appearance of small, ring-shaped necrotic spots associated with hypersensitivity reactions. These necrotic symptoms do not impair the development of pumpkin plants infected with PRSV or those possessing the PRSV-resistant gene. For example, the combination with the recessive ZYMV-resistant gene, which is already widely used in commercially available hybrid pumpkins and described in International Publication No. 2012 / 143391, provides a high level of resistance to PRSV and is sufficient to prevent any symptoms in the leaves and fruits.

[0043] The terms "hybrid," "hybrid plant," and "hybrid offspring" refer to individuals produced from genetically different parents (for example, individuals that are genetically heterozygous or nearly heterozygous).

[0044] The term "inbred line" refers to a population that is genetically homozygous or nearly homozygous. Inbred lines can be obtained, for example, through several cycles of sibling breeding or self-breeding, or by diploid haploid generation.

[0045] The term "diploid haploid" refers to a stable inbred line derived from another culture. Several pollen grains (haploids) cultivated in a specific culture medium and environment can develop embryos containing n chromosomes. These small plants are then "diploidized," containing 2n chromosomes. The offspring of these plants are named "diploid haploids" and are virtually no longer segregated (stable).

[0046] The term "genetically fixed" refers to a gene sequence that is stably incorporated into the genome of a plant that does not normally contain the gene sequence. When genetically fixed, the gene sequence can be easily and predictably inherited by other plants through sexual mating.

[0047] The terms “plant body” or “plant part” mean, in the following context, any part, organ, or tissue of a plant that can be obtained from a pumpkin plant body according to the present invention, for example, but not limited to, leaves, stems, roots, flowers or flower parts, fruits, shoots, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic regions, callus tissue, seeds, cuts, cells or tissue cultures, or any other part or product of a plant body that, when grown into a fruit-producing plant body, still exhibits the improved papaya earring spot virus resistance trait according to the present invention.

[0048] A "plant body" is any plant body at any stage of development.

[0049] Pumpkin plant seeds are seeds that grow into pumpkin plants according to any of the embodiments.

[0050] A "plant cell" is the structural and physiological unit of a plant body, including the protoplast and cell wall. Plant cells can exist as isolated single cells or cultured cells, or as part of a highly organized unit, such as plant tissue, plant organs, or a whole plant.

[0051] "Plant cell cultures" refer to cultures of plant units such as protoplasts at various developmental stages, cultured cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos.

[0052] "Plant organs" are clearly and visibly structured and differentiated parts of a plant body, such as roots, stems, leaves, buds, or embryos.

[0053] As used herein, “plant tissue” means a group of plant cells organized into structural and functional units. This includes any tissue of a plant in an implanter or under culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. When the term is used in combination with or without any particular type of plant tissue listed above or included in this definition, it is not intended to exclude any other type of plant tissue.

[0054] As used herein, the term “breeding” and its grammatical variations refer to any process that produces offspring individuals. Breeding can be sexual, asexual, or any combination thereof. Exemplary and non-limiting types of breeding include crossbreeding, self-pollination, derivative production of doubling haploids, and combinations thereof.

[0055] As used herein, the term “established breeding population” refers to a collection of potential breeding material produced by and / or used as parents in a breeding program, e.g., a commercial breeding program. Members of an established breeding population are typically well-characterized genetically and / or phenotypic. For example, several phenotypic traits of interest may be evaluated, e.g., under different environmental conditions, at multiple locations, and / or at different time points. Alternatively or in addition, one or more loci associated with the expression of a phenotypic trait may be identified, and one or more members of the breeding population may be genotyped with respect to one or more loci and one or more genetic markers associated with one or more loci.

[0056] As used herein, the term “diploid individual” refers to an individual having two sets of chromosomes, typically one from each of its two parents. However, in some embodiments, it is understood that a diploid individual may inherit its “mother” and “father” sets of chromosomes from the same single organism, for example, when a plant self-pollinates to produce the next generation of plant bodies.

[0057] In the scope of this invention, "homozygous" is understood to refer to similar alleles at one or more corresponding loci on homologous chromosomes. In relation to this invention, a pumpkin plant containing two identical copies of a particular gene transfer sequence at a particular locus, for example, a gene transfer sequence located on chromosome 10, is homozygous at the corresponding locus.

[0058] Within the scope of this invention, "heterozygous" is understood to refer to different alleles at one or more corresponding loci on homologous chromosomes. In relation to this invention, a pumpkin plant containing one copy of a specific gene transfer sequence at a particular locus, for example, a gene transfer sequence located on chromosome 10, is heterozygous at the corresponding locus.

[0059] Within the scope of this invention, the "dominant" allele is understood to refer to the allele that determines the phenotype when present in a heterozygous or homozygous state.

[0060] Within the scope of this invention, a "semi-dominant" allele is understood to refer to an allele that determines the phenotype when present in a heterozygous or homozygous state. However, the strength of the phenotype is generally higher when the allele is present in a homozygous state.

[0061] A "recessive" allele refers to an allele that determines the phenotype only when it is present in a homozygous state.

[0062] Within the scope of this invention, "backcrossing" is understood to refer to the process of repeatedly crossing the offspring of a hybrid back with one of its parents. Different recurrent parents may be used in subsequent backcrosses.

[0063] Within the scope of this invention, "locus" is understood to refer to a region on a chromosome that contains a gene, an allele, or a corresponding gene sequence that contributes to a trait.

[0064] As used herein, “marker locus” means a region on a chromosome that is present in the genome of an individual and contains a nucleotide or polynucleotide sequence associated with one or more loci of interest, which may include any other genetic determinants or factors that contribute to a gene or trait.

[0065] Within the scope of this invention, "genetic linkage" is understood to refer to the relationship between traits in inheritance based on the location of adjacent genes on the same chromosome, as measured by the recombination rate between gene loci (centimorgan, cM).

[0066] In relation to the subject matter of this disclosure, as used herein, the terms “sexual mating” and “sexual reproduction” refer to the production of offspring by the fusion of gametes (for example, by fertilization, such as pollination to produce seeds in a plant). “Sexual mating” or “cross-fertilization” refers, in some embodiments, to the fertilization of one individual by another individual (for example, cross-pollination in a plant). The term “self-pollination” refers, in some embodiments, to the production of seeds by self-fertilization or self-pollination, i.e., the pollen and ovules coming from the same plant.

[0067] As used herein, the terms “genetic marker” or “DNA marker” refer to genomic features of an individual associated with one or more loci of interest (e.g., nucleotide or polynucleotide sequences present in the individual’s genome). In some embodiments, a genetic marker is, depending on the context, a polymorphism or a locus occupied by a polymorphism in the population of interest. Many other examples of genetic markers include, for example, single nucleotide polymorphisms (SNPs), indels (i.e., insertions / deletions), simple repeat sequences (SSRs), restriction fragment length polymorphisms (RFLPs), randomly amplified polymorphic DNA (RAPDs), cleavage-amplified polymorphic sequence (CAPS) markers, diversity array technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs). Genetic markers may be used, for example, to locate loci containing alleles on a chromosome that contribute to phenotypic variability. The term “genetic marker” may also refer to polynucleotide sequences complementary to a genomic sequence, such as a nucleic acid sequence used as a probe.

[0068] As used herein, the term “genotype” refers to the genetic makeup of a cell or organism. “Genotype for a set of genetic markers” of an individual includes specific alleles for one or more genetic marker loci present in the individual’s haplotype.

[0069] As used herein, the term “offspring” refers to the offspring of a particular cross. Typically, offspring result from the breeding of two individuals, although some species (in particular some plants and hermaphroditic animals) can self-pollinate (i.e., the same plant body acts as a donor for both male and female gametes). Offspring may be, for example, F1, F2, or any subsequent generation.

[0070] As used herein, the term “quantitative trait locus” (QTL) refers to an association between a genetic marker and a chromosomal region and / or gene and / or gene transfer sequence that influences the phenotype of a trait of interest. Typically, this is determined statistically, for example, based on one or more methods published in the literature. A QTL may be a chromosomal region and / or locus containing at least two alleles that influence a phenotypic trait in a different way.

[0071] The term "recipient pumpkin plant" is used herein to refer to a pumpkin plant that receives DNA obtained from a donor pumpkin plant containing a gene transfer sequence related to improved papaya ringspot virus resistance.

[0072] The term “natural genetic background” is used herein to refer to the original genetic background of a gene sequence. For example, the gene sequence of the present invention was found at a specific location on chromosome 10 of the Cucurbita ecuadorensis plant. Conversely, a method involving the introduction of DNA containing this gene sequence, derived from chromosome 10 of the Cucurbita ecuadorensis plant, to the same location on chromosome 10 of another pumpkin species, preferably a cultivated pumpkin plant, and more preferably a C. pepo subsp. pepo var. cylindrica plant, for example through breeding, would result in this gene sequence not being present in the natural genetic background. When the gene sequences of the present invention are introduced from a Cucurbita ecuadorensis background into another pumpkin species, preferably a cultivated pumpkin plant, and more preferably into a C. pepo subsp. pepo var. cylindrica plant, they are referred to as "introduced gene sequences" or "introduced gene sequences."

[0073] Within the scope of this invention, "donor pumpkin plant" is understood to mean a pumpkin plant that provides a gene transfer sequence related to improved papaya ringspot virus resistance.

[0074] Within the scope of the present invention, “marker-based selection” is understood to mean, for example, the use of genetic markers for detecting one or more nucleic acids of plant origin, which can be used (or avoided) in a selective breeding program to identify plants that have alleles relating to desired (or undesired) traits in relation to desired traits.

[0075] Single nucleotide polymorphisms (SNPs), which are mutations at a single site in DNA, are the most common type of mutation in the genome. SNPs are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome (or other covalent sequence) differs between members of a species or between paired chromosomes in an individual. For example, two sequenced DNA fragments from different individuals, AAGCCTA vs. AAGCTTA, contain a single nucleotide difference. In this case, there are two alleles: C and T. The basic principles of SNP arrays are the same as those of DNA microarrays. These are DNA hybridization, fluorescence microscopy, and DNA capture convergence. The three components of an SNP array are an array containing nucleic acid sequences (i.e., amplified sequences or targets), one or more labeled allele-specific oligonucleotide probes, and a detection system for recording and interpreting the hybridization signal. The presence or absence of a desired SNP marker can be determined by real-time PCR using double-stranded DNA dyes or fluorescent reporter probes.

[0076] Within the scope of this invention, "PCR (polymerase chain reaction)" is understood to refer to a method that enables various analyses based on a specific region of DNA or a subset of the genome by generating a relatively large amount of that region. Within the scope of this invention, "PCR primer" is understood to refer to a relatively short fragment of single-stranded DNA used for PCR amplification of a specific region of DNA.

[0077] As used herein, "probe" refers to a group of atoms or molecules that can recognize and bind to a specific target molecule or cellular structure, thereby enabling the detection of that target molecule or structure. In particular, "probe" refers to a labeled DNA or RNA sequence that can be used to detect and quantify the presence of a complementary sequence by molecular hybridization.

[0078] "Sequence Identity". The terms "identical" or "identical" relating to two or more nucleic acid or protein sequences refer to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides when compared and aligned for the greatest match, as measured by one of the following sequence comparison algorithms or by visual inspection. If the lengths of the two sequences to be compared are different, sequence identity preferably relates to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues in the longer sequence. As used herein, the identity / homology percentage between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., identity % = number of identical positions / total number of positions × 100). Sequence comparison and determination of the identity percentage between two sequences can be achieved using mathematical algorithms, as described later herein. For example, sequence identity can be determined using a computer program such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711), as in the conventional method. Bestfit utilizes the locus homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to find the segment with the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has 95% identity with, for example, the reference sequence of the present invention, the parameters are preferably adjusted so that the percentage of identity is calculated over the entire length of the reference sequence and a homology gap of up to 5% of the total number of nucleotides in the reference sequence is permitted.When using Bestfit, so-called arbitrary parameters are preferably left at their preset ("initial") values. Deviations observed in the comparison between a given sequence and the sequences of the present invention may be caused, for example, by additions, deletions, substitutions, insertions, or recombinations. Such sequence comparisons may also be performed using the program "fasta20u66" (version 2.0u66 by William R. Pearson and the University of Virginia, September 1998; see also WRPearson (1990), Methods in Enzymology 183, 63-98, attached examples and http: / / workbench.sdsc.edu / ). For this purpose, "default" parameter settings may be used.

[0079] Embodiment Plant body, seeds, fruits. In the first embodiment, the present invention relates to a cultivated pumpkin plant resistant to papaya earring spot virus (PRSV) infection, preferably a cultivated Cucurbita pepo plant, wherein its genome contains a gene transfer sequence derived from Cucurbita ecuadorensis that confers resistance to PRSV, the gene transfer sequence being located on chromosome 10 and containing the following SNP markers: a) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; b) A genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of SEQ ID NO: 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) A genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; g) A C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) A C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for SNP marker 11 at position 36 of SEQ ID NO: 11; and / or l) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12. The present invention provides a plant body containing at least one of the following.

[0080] In a further embodiment of the present invention, the gene transfer sequence includes at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and / or SEQ ID NO: 12, or at least one of the sequences that is at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% identical to one or more of the aforementioned sequences.

[0081] In a further embodiment of the present invention, the plant body includes the SNP marker 4.

[0082] In a further embodiment of the present invention, the plant is heterozygous for the at least one SNP marker. In a further embodiment, the plant is homozygous for the at least one SNP marker.

[0083] In a further embodiment of the present invention, the plant of any of the above embodiments further comprises a Zn allele located on chromosome 16 that is associated with zucchini yellow mosaic virus (ZYMV) resistance, wherein the Zn allele comprises a G genotype that is homozygous for SNP marker 13 at position 119 of SEQ ID NO: 13.

[0084] In further embodiments, the gene transfer sequence is contained in the Cucurbita pepo plant 22SQE800260 (whose representative seed is deposited under NCIMB acceptance number 44148), or in its offspring or ancestors.

[0085] In a further embodiment, the present invention provides a plant according to any of the above embodiments, which is obtained by crossing a Cucurbita pepo plant 22SQE800260, whose representative seeds are deposited under NCIMB accession number 44148, or its offspring or ancestor, with a pumpkin plant that does not contain the gene transfer sequence that confers PRSV resistance.

[0086] In a further embodiment, the present invention provides a plant according to any of the above embodiments, wherein the plant is an inbred, diploid haploid, diploid, or hybrid plant.

[0087] In another embodiment, the plant according to the present invention is male-sterile. In yet another embodiment, the plant according to the present invention is cytoplasmically male-sterile.

[0088] In further embodiments, the pumpkin plant of the present invention is a pumpkin plant according to any of the embodiments described above, where the PRSV resistance-constituting gene transfer sequence located on chromosome 10 can be identified using any of the SNP markers 1 to 12 disclosed in Table 5 below.

[0089] In further embodiments, the present invention provides a cultivated pumpkin plant resistant to PRSV, preferably a cultivated C. pepo plant, more preferably a cultivated C. pepo subsp. pepo plant, and even more preferably a cultivated C. pepo subsp. pepo var. cylindrica plant, wherein its genome contains a gene transfer sequence located on chromosome 10, derived from a Cucurbita ecuadorensis plant, which confers resistance to PRSV, and wherein the plant genome is a) Sequence ID 1, and b) Sequence ID 12 including

[0090] In a further embodiment, the cultivated pumpkin plant of the above embodiment further includes at least a third SNP marker from the list of SNP markers 2 to 11 disclosed in Table 5.

[0091] In further embodiments, the present invention relates to a PRSV-resistant cultivated pumpkin plant, preferably a cultivated C. pepo plant, more preferably a cultivated C. pepo subsp. pepo plant, and even more preferably a cultivated C. pepo subsp. pepo var. cylindrica, wherein its genome contains a gene transfer sequence located on chromosome 10, derived from a Cucurbita ecuadorensis plant, which confers resistance to papaya ringspot virus, and the plant genome is a) G genotypes that are heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of Sequence ID No. 4. We provide plants that include [this].

[0092] In further embodiments, the pumpkin plant of the present invention is a pumpkin plant according to any of the embodiments described above, the representative seed of which is deposited under NCIMB accession number 44148, the Cucurbita pepo plant 22SQE800260 or its offspring or ancestors, is the source of the PRSV resistance-conferring gene transfer sequence.

[0093] A further embodiment of the present invention is to provide a plant portion of a plant body according to any one of the embodiments described above.

[0094] A further embodiment provides any other part or product of a plant that still exhibits the papaya earring spot virus resistance trait according to the present invention when grown into a plant that produces fruit, such as, but is not limited to, leaves, stems, roots, flowers or flower parts, fruits, shoots, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic regions, callus tissue, seeds, cuts, cells or tissue cultures, or in particular a plant that produces fruit.

[0095] In a further embodiment, the present invention provides seeds for producing plants according to any of the embodiments described above.

[0096] In further embodiments, the present invention relates to the use of a pumpkin plant according to any of the above embodiments as a rootstock, preferably a rootstock for a cucurbitaceae plant, more preferably a rootstock for a pumpkin. In further embodiments, the present invention relates to the use of a Cucurbita pepo plant 22SQE800260 or its offspring or ancestors, whose representative seeds are deposited under NCIMB accession number 44148, as a pumpkin rootstock.

[0097] In another embodiment, the use of a pumpkin plant, plant part, or seed according to any of the embodiments described above for producing and harvesting pumpkin fruits is envisioned.

[0098] In another embodiment, the present invention relates to the use of a pumpkin plant, plant part, or seed according to any embodiment, wherein the pumpkin plant, plant part, or seed is a Cucurbita pepo plant 22SQE800260 or its offspring or ancestor, the representative seed of which is deposited under NCIMB accession number 44148.

[0099] In further embodiments, the present invention relates to the use of pumpkin plants, plant parts, or seeds according to any of the embodiments described above for sowing in fields, greenhouses, or plastic greenhouses.

[0100] In one embodiment, the present invention provides a pumpkin fruit produced by a pumpkin plant according to any of the embodiments described above.

[0101] The present invention further relates to the use of pumpkin plants according to any of the above embodiments for gene transfer of the PRSV resistance trait into pumpkin plants lacking the PRSV resistance trait.

[0102] Genetic sequences, markers. The present invention further relates to a gene transfer sequence associated with PRSV resistance traits in pumpkin plants. In a further embodiment, the gene sequence of the present invention is located on chromosome 10. In a further embodiment of the present invention, the gene sequence is contained in, obtained from, or can be obtained from the Cucurbita pepo plant 22SQE800260, whose representative seeds are deposited under NCIMB accession number 44148 and which contains the gene sequence, or its offspring or ancestors.

[0103] In another embodiment, the gene transfer sequence of the present invention is located on chromosome 10 and has the following SNP markers: a) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; b) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of SEQ ID NO: 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; g) A C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) A C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for SNP marker 11 at position 36 of SEQ ID NO: 11; and / or l) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12. Characterized by at least one of the following:

[0104] The present invention also discloses the use of at least one, at least two, or at least three SNP markers according to the present invention for the diagnostic selection and / or genotyping of PRSV resistance trait loci in pumpkin plants, particularly cultivated pumpkin plants.

[0105] The present invention further discloses the use of at least one, at least two, or at least three SNP markers according to the present invention to identify the presence of PRSV resistance traits in pumpkin plants, particularly cultivated pumpkin plants, and more particularly in pumpkin plants according to the present invention, and / or to monitor gene transfer of PRSV resistance traits in pumpkin plants, particularly cultivated pumpkin plants, and particularly in pumpkin plants according to the present invention as described herein.

[0106] Accordingly, in one embodiment, the present invention further relates to a derived marker, in particular a derived primer or probe, developed from the amplification product according to this specification by methods known in the art, as described above, wherein the derived marker is genetically associated with the PRSV resistance trait locus.

[0107] Breeding methods. In further embodiments, the present invention relates to a method for producing cultivated pumpkin plants, preferably Cucurbita pepo plants, that are resistant to PRSV infection. a) A step of crossing a plant according to any one of claims 1 to 7 with a cultivated pumpkin plant lacking the gene transfer sequence that confers PRSV resistance, b) A step of selecting a progeny plant containing the gene transfer sequence located on chromosome 10 that confers resistance to PRSV, the following SNP marker: i) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; ii) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of sequence number 2; iii) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; iv) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; v) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; vi) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; vii) A C genotype that is heterozygous or homozygous for SNP marker 7 at position 36 of sequence number 7; viii) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; ix) A C genotype that is heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; x) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; xi) A genotype that is heterozygous or homozygous for SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or xii) A genotype that is heterozygous or homozygous for SNP marker 12 at position 36 of sequence number 12. A selection step including detecting at least one of the following The present invention provides a method for producing plants that contain and thereby enhance resistance to PRSV.

[0108] In further embodiments, the present invention relates to the method according to the embodiments described above, c) Propagating the selected offspring or crossbreeding the selected offspring with another pumpkin plant to produce further offspring. This provides a method that further includes this.

[0109] In further embodiments, the present invention provides a method according to the above-described embodiments for selecting further offspring and allowing them to be self-bred / crossed for a further 2 to 10 generations.

[0110] In a further embodiment, the present invention provides a method according to the above-described embodiment, further comprising the step of detecting a G genotype that is heterozygous or homozygous for the SNP marker 13 at the position corresponding to position 119 of SEQ ID NO: 13.

[0111] In further embodiments, the present invention provides a method according to the above embodiments, wherein the plant in step a) is Cucurbita pepo plant 22SQE800260, whose representative seeds are deposited under NCIMB accession number 44148, or a descendant or ancestor thereof.

[0112] In another embodiment, the present invention relates to a method for producing a plant body, plant part, or seed of a PRSV-resistant pumpkin, comprising the following steps: a) A step of crossing a first plant lacking the gene transfer sequence that confers PRSV resistance according to the present invention with a second pumpkin plant according to any embodiment, b) Steps to obtain offspring pumpkin plants, c) Optionally, the step of selecting a descendant plant characterized as exhibiting resistance to PRSV. Regarding methods including

[0113] In further embodiments, the present invention relates to a method according to the above-described embodiments, wherein the second pumpkin plant is Cucurbita pepo plant 22SQE800260, whose representative seeds are deposited under NCIMB accession number 44148, or a descendant or ancestor thereof.

[0114] In another embodiment, the present invention involves the following steps: a) A step of preparing seeds of a pumpkin plant according to any of the embodiments described above, b) The step of sowing the aforementioned seeds and then growing mature, fertile plants, c)a) The step of inducing self-pollination of the plant body, growing the fruit, and then harvesting fertile seeds, d)c) The step of growing plants from the harvested seeds and selecting pumpkin plants resistant to Papa Earrings Spot Virus and This relates to a method for producing PRSV-resistant pumpkin plants, including [specific example].

[0115] In another embodiment, the present invention provides a method for increasing the resistance of pumpkin plants to PRSV, comprising the following steps: a) A step of selecting pumpkins containing a PRSV-resistant trait associated with a gene transfer sequence located on chromosome 10, wherein the trait can be identified by the presence of at least one of the SNP markers listed in Table 5; b) A step of crossing the plant body from step a) containing the PRSV resistance trait with a pumpkin plant that does not contain the PRSV resistance trait and is sensitive to PRSV, particularly a cultivated pumpkin plant, compared to the plant body from step a), c) A step of selecting offspring from the cross that show increased PRSV resistance compared to the plant in step b), and also show the ZYMV resistance gene if it is not present in the second parent. Regarding methods including

[0116] In further embodiments, the present invention relates to a method according to the above-described embodiments, wherein the plant in step a) also includes a ZYMV resistance trait conferred by a sequence located on chromosome 16, which can be identified by the SNP markers listed in Table 8.

[0117] In further embodiments, the present invention provides a method for producing F1 pumpkin plants resistant to PRSV, comprising crossing an inbred pumpkin plant, which is a plant according to any one of the above embodiments, with a different inbred pumpkin plant to produce F1 hybrid offspring.

[0118] Selection method. In further embodiments, the present invention relates to cultivated pumpkin plants, preferably cultivated C. pepo plants, more preferably cultivated C. pepo subsp. pepo plants, and even more preferably cultivated C. pepo subsp. pepo var. cylindrica plants, which exhibit resistance to PRSV infection and have at least one copy of a gene transfer sequence that confers the PRSV resistance, and which have the following SNP markers: a) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; b) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of SEQ ID NO: 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; g) A C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) A C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for SNP marker 11 at position 36 of SEQ ID NO: 11; and / or l) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12. The present invention provides a method for identifying pumpkin plants that exhibit resistance to PRSV, comprising the step of detecting at least one of the following.

[0119] In further embodiments, the present invention provides a method according to the above-described embodiments, further comprising selecting a pumpkin plant containing one or more SNP markers, and crossing the selected pumpkin plant with a second pumpkin plant to produce offspring pumpkin plants containing at least one of the SNP markers and exhibiting resistance to PRSV.

[0120] In further embodiments, the present invention provides a method for producing pumpkin seeds, comprising growing a pumpkin plant from the seeds of the above embodiments and causing the plant to produce further pumpkin seeds.

[0121] In another embodiment, the present invention relates to a method for identifying pumpkin plants containing the PRSV resistance-conferring gene transfer sequence of the present invention, a) A step of preparing a population from which the PRSV resistance trait is isolated, b) A step of screening a segregated population for members exhibiting resistance to PRSV, wherein the trait can be identified by the presence of the PRSV resistance-constituting gene transfer sequence of the present invention; c) A step of selecting one member of a segregated population, wherein the member includes the PRSV resistance trait. This provides a method that includes this.

[0122] In a further embodiment, the present invention relates to a method for identifying a cultivated pumpkin plant having a gene transfer sequence on chromosome 10, wherein the gene transfer sequence confers resistance to PRSV. a) To provide a population that can be separated for PRSV resistance, b) Screening the population using a kit that detects at least one of the SNP markers listed in Table 5, c) Identifying a plant containing at least one of the SNP markers selected from the list in Table 5. This provides a method that includes this.

[0123] In further embodiments, the present invention relates to a method for identifying a pumpkin source with a PRSV-resistant trait on chromosome 10, a) To provide a pumpkin variety or multiple pumpkin varieties, b) Screening the pumpkin strain or multiple pumpkin strains using a kit that detects at least one of the SNP markers listed in Table 5, c) Identify a wild pumpkin strain containing at least one of the SNP markers selected from the list in Table 5. This provides a method that includes this.

[0124] In yet another embodiment, the present invention relates to the use of at least one SNP marker amplified from the genome of a pumpkin plant according to any of the above embodiments, preferably from the genome of Cucurbita pepo plant 22SQE800260 or its offspring or ancestor, whose representative seeds are deposited under NCIMB accession number 44148, wherein the SNP marker is identified using one of the SNP markers listed in Table 5, and the SNP marker indicates the presence of a PRSV-resistant trait in the pumpkin plant, thereby identifying a pumpkin plant that contains and exhibits the PRSV-resistant trait.

[0125] In further embodiments, the present invention relates to a method for evaluating the genotype of cultivated pumpkin plants that exhibit resistance to PRSV, preferably cultivated Cucurbita pepo plants, more preferably cultivated C. pepo subsp. pepo plants, and even more preferably cultivated C. pepo subsp. pepo var. cylindrica plants. a) A step of preparing a sample from the plant body, b) A step of detecting a QTL locus located on chromosome 10 and associated with PRSV resistance in the sample, wherein the QTL locus is identified by the following SNP marker: i) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; ii) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of sequence number 2; iii) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; iv) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; v) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; vi) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; vii) A C genotype that is heterozygous or homozygous for SNP marker 7 at position 36 of sequence number 7; viii) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; ix) A C genotype that is heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; x) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; xi) A genotype that is heterozygous or homozygous for SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or xii) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12; and / or xiii) Any other DNA marker associated with the QTL locus, flanked by SNP markers 1 and 12 A step including at least one of the following This provides a method that includes this.

[0126] In a further embodiment, the present invention relates to a method according to the above-described embodiment, further comprising the step of detecting a ZYMV resistance trait conferred in the plant by a sequence located on chromosome 16, wherein the QTL comprises a G genotype that is homozygous for SNP marker 13 at the position corresponding to position 119 of sequence number 13.

[0127] In further embodiments, the present invention provides a method for identifying gene transfer sequences associated with high resistance to PRSV in cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, more preferably cultivated C. pepo subsp. pepo plants, and even more preferably cultivated C. pepo subsp. pepo var. cylindrica plants, comprising the step of detecting an allele of at least one DNA marker genetically associated with a QTL locus associated with high resistance to PRSV in the plant, wherein the allele is located within 10 cM, preferably within 5 cM, of the QTL locus located on chromosome 10 in a genomic region flanked by SNP markers 1 and 12.

[0128] In further embodiments, the present invention relates to the method according to the above-described embodiments, wherein the QTL locus is identified by the following SNP markers: a) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of Sequence ID No. 1; b) A G genotype that is heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of SEQ ID NO: 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) A G genotype that is heterozygous or homozygous for SNP marker 6 at the position corresponding to position 36 of sequence number 6; g) A C genotype that is heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) A C genotype that is heterozygous or homozygous for SNP marker 9 at the position corresponding to position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for SNP marker 11 at position 36 of SEQ ID NO: 11; and / or l) A genotype that is heterozygous or homozygous for SNP marker 12 at the position corresponding to position 36 of sequence number 12. The present invention provides a method that can be identified by at least one of the following.

[0129] In further embodiments, the present invention provides a method according to the above embodiments, comprising the step of selecting a cultivated pumpkin plant containing the gene transfer sequence, preferably a cultivated Cucurbita pepo plant, more preferably a cultivated C. pepo subsp. pepo plant, and even more preferably a cultivated C. pepo subsp. pepo var. cylindrica plant.

[0130] In further embodiments, the present invention relates to a method according to any of the embodiments described above, further comprising the step of detecting in the plant a ZYMV resistance trait conferred by a sequence located on chromosome 16, which can be identified by the SNP markers listed in Table 8.

[0131] In a further embodiment, the present invention provides a method for identifying cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, that exhibit high resistance to PRSV by identifying QTLs associated with high resistance to PRSV, a) A step of detecting at least one DNA marker derived from a pumpkin plant (this DNA marker is linked to a chromosomal segment associated with high resistance to PRSV), wherein the chromosomal segment is flanked on both sides by SNP markers having at least 80% sequence identity to sequence numbers 1 and 12; b) The step of identifying the pumpkin plant containing the at least one DNA marker. This provides a method that includes [something].

[0132] In further embodiments, the present invention relates to a method for identifying a wild pumpkin source with a PRSV-resistant trait on chromosome 10, a) To provide one or more wild pumpkin varieties; b) Screening one or more wild pumpkin strains using a kit that detects at least one of the SNP markers listed in Table 5, c) Identify a wild pumpkin strain containing at least one of the SNP markers selected from the list in Table 5. This provides a method that includes [something].

[0133] In a further embodiment, the present invention provides the use of any of Sequence IDs 1 to 12 for screening a population of pumpkin plants for the presence of a QTL locus located on chromosome 10 that is associated with high PRSV resistance.

[0134] use. The present invention also relates to the use of PRSV-resistant propagating material, which can be obtained from pumpkin plants according to any of the above embodiments, for growing pumpkin plants to produce PRSV-resistant pumpkin plants, wherein the PRSV resistance can be evaluated by a standard assay, in particular the assay described in Example 2 below.

[0135] The present invention also relates to the use of PRSV-resistant propagation material that can be obtained from pumpkin plants according to any of the aforementioned embodiments for producing pumpkin fruits.

[0136] In another embodiment, the present invention relates to the use of cultivated pumpkin plants, plant parts, or seeds, more preferably cultivated C. pepo subsp. pepo var. cylindrical, for growing plants and producing and harvesting crops and / or fruits, according to any of the above embodiments.

[0137] In another embodiment, the present invention relates to the use of cultivated pumpkin plants, more preferably cultivated C. pepo subsp. pepo var. cylindrical, according to any of the above embodiments, for producing fruit for fresh markets or food processing.

[0138] In another embodiment, the present invention relates to the use of cultivated pumpkin plants, plant parts, or seeds, preferably cultivated C. pepo subsp. pepo var. cylindrical plants, plant parts, or seeds, wherein the cultivated pumpkin plants, plant parts, or seeds, preferably cultivated C. pepo subsp. pepo var. cylindrical plants, plant parts, or seeds, are of Cucurbita pepo plant 22SQE800260 (its representative seeds are deposited under NCIMB accession number 44148), or its descendants or ancestors.

[0139] In further embodiments, the present invention relates to the use of cultivated pumpkin plants, plant parts, or seeds, more preferably cultivated C. pepo subsp. pepo var. cylindrical, for sowing in fields, greenhouses, or plastic greenhouses, according to any of the above embodiments.

[0140] In further embodiments, the present invention relates to the use of pumpkin plants according to any of the above embodiments for conferring a high PRSV resistance trait to pumpkin plants lacking the trait. The present invention further relates to the use of pumpkin plants according to any of the above embodiments for gene transfer of a high PRSV resistance trait into pumpkin plants lacking the trait.

[0141] In a further embodiment, the present invention provides the use of any of Sequence IDs 1 to 12 for screening a population of pumpkin plants for the presence of a QTL locus located on chromosome 10 that is associated with high PRSV resistance.

[0142] In a further embodiment, the present invention relates to the use of Sequence ID No. 4 for screening a population of pumpkin plants for the presence of a QTL locus located on chromosome 10 and associated with high PRSV resistance.

[0143] Based on the description of the present invention, any person skilled in the art who possesses a representative seed of Cucurbita pepo plant 22SQE800260, deposited under NCIMB accession number 44148, containing the gene transfer sequence described herein, or its offspring, will not encounter difficulty introducing the gene transfer sequence of the present invention into various types of other pumpkin plants using breeding techniques well known in the art, with the support of the SNP markers disclosed herein.

[0144] Details of seed deposit On May 10, 2023, the applicant deposited 625 seeds of Cucurbita pepo plant 22SQE800260 with NCIMB (National Collection of Industrial, Food and Marine Bacteria (NCIMB) Limited, Wellheads Place, Dyce, Aberdeen, AB21 7GB) under NCIMB accession number 44148.

[0145] The applicant shall select an expert solution and, until the public notice of the grant of this patent is published, or if this application is rejected, withdrawn, or deemed withdrawn, for a period of 20 years from the filing date, the deposited material shall be made available only to experts in accordance with EPC Rule 32(1) or the corresponding laws or treaties of other countries (expert witness provisions).

[0146] Cucurbita pepo hybrid plant 22SQE800260 is homozygous for the PRSV resistance QTR on chromosome 10, meaning that Cucurbita pepo hybrid plant 22SQE800260 contains one copy of the gene transfer sequence associated with high PRSV resistance on chromosome 10.

[0147] Cucurbita pepo hybrid plant 22SQE800260 also contains two copies of a recessive ZYMV-resistant QTL allele located on chromosome 16. [Examples]

[0148] Example 1: Development of germplasm and population The Cucurbita ecuadorensis strain SQE0000542 was confirmed to exhibit high resistance to Potivirus and PRSV in our internal breeding germplasm, and was therefore used as a gene source for PRSV trait transfer to green zucchini, white pumpkin, and gray pumpkin varieties.

[0149] To avoid pollen incompatibility issues arising from crosses between Cucurbita ecuadorensis and the green cylindrical line, a backcross population was developed using embryo rescue. In each backcross generation, the material was self-pollinated, selected based on resistance phenotypes, and then subjected to another backcross cycle up to the BC5F5 generation.

[0150] This line was crossed with a green cylindrical line that lacked any resistance factors to potivirus to develop F2-F3 populations. F2 plants were sampled, their genotypes determined using 200 markers, and they were individually self-pollinated. Under controlled conditions in a growth chamber, the plants were inoculated with Papa Earlingspot virus at the cotyledon stage, and the phenotypes of 173 F3 families were analyzed, with each individual plant evaluated on a scale of 1 to 9.

[0151] Example 2: Protocol. Example 2A. Papa Earring Spot Virus Inoculation Material For long-term storage at -80°C, papaya earring spot virus (PRSV) isolates were maintained on frozen leaf tissue. PRSV was preserved by collecting several young infected leaves from the original infection batch and storing them in aluminum foil bags in 5-gram or multiples of 5-gram portions in a freezer. For propagation and before proceeding to large-scale testing and inoculation, dilution tests were performed to measure the charge of the virus. Depending on the virus concentration, inoculation volume, and dilution ratio required for the test, the amount of isolate was calculated according to the following rules shown in Table 1. Inoculation material was prepared according to the steps described below. The pH of the cold buffer (pre-stored in the refrigerator) was verified as neutral and corrected as necessary. Frozen infected leaf samples were ground in a mortar that had been pre-autoclaved and maintained under cold conditions on ice. A portion of the desired volume of buffer solution was then added and mixed with the leaves. Once the mixture was homogenized, the remainder of the buffer solution was added, followed by coal and carborundum, and thoroughly mixed.

[0152] [Table 1]

[0153] The initial amount of isolate used for preparation depends on the amount required for the inoculation material, and since the batch is made of 5 grams of leaves, the fraction is rounded up to 5 grams. Coal was used to color the inoculation material and confirm that it was a proper inoculation, while carborundum was used for its abrasive properties to scratch the cotyledons and allow for viral infection.

[0154] Example 2B. Preparation and inoculation of plant material. F3 families were evaluated for PRSV resistance using artificial inoculation. F3 seedlings from 173 crosses between a resistant source and a susceptible green zucchini (Cucurbita pepo) line were sown in trays containing a compost mixture. Each tray was filled with three F3 families: eight seedlings from the resistant parent (14SQE150025), eight seedlings from the susceptible parent (15SQE310133), and 24 seedlings. Eight days after sowing, at the cotyledon stage, the plants were inoculated with the PRSV inoculum prepared as described above. The mixture was rubbed twice by hand onto each cotyledon to allow the inoculum to penetrate the tissue.

[0155] Example 2C. Scoring of PRSV resistance. The initial symptoms, leaf yellowing and necrotic ring-shaped spots and lesions, appeared 10–14 days (dpi) after inoculation. Plants were monitored, and symptoms were evaluated at 14, 21, and 28 dpi. Plants were scored on a quantitative scale as described below, and are illustrated in Figure 1.

[0156] Evaluation Symptoms 9. A healthy plant with no symptoms. 8. The diseased area covers 1% to 5% of the leaves. 7. The affected area covers 5% to 15% of the leaf; the yellowed parts or spots on the leaf are limited. 6. The diseased area covers 16% to 30% of the leaf. 5. The lesion area covers 31%–45% of the leaf; there is significant but moderate yellowing and small vein banding. 4. The diseased area covers 46% to 60% of the leaf. 3. The lesion area covers 61% to 75% of the leaf; vein banding, dense yellowing areas. 2. The diseased area covers 76% to 90% of the leaf. 1. The affected area covers 91% to 100% of the leaf; severe symptoms and leaf yellowing, severe vein banding and marking of yellowed veins, leaf curling.

[0157] All plants were scored using the above semi-quantitative evaluation scale (1-9). The disease score for each strain was calculated using the following calculations, along with the individual plant scores and the strain-adjusted average. Score = ((R×9)+(S×8)+(T×7)+(U×6)+(V×5)+(W×4)+(X×3)+(Y×2)+(Z×1)) / R+S+T+U+V+W+X+Y+Z; in the formula The number of plants with a score equal to R=9; The number of plants with a score equal to S=8; The number of plants with a score equal to T=7; The number of plants with a score equal to U=6; The number of plants with a score equal to V=5; The number of plants with a score equal to W=4; The number of plants with a score equal to X=3; The number of plants with a score equal to Y=2; and The number of plants with a score equal to Z=1.

[0158] Example 2D. Method for identifying QTLs and corresponding gene transfer sequences that underlie the PRSV high resistance trait. To discover QTLs, 180 individual plants from an F2 population inherited from a cross between the resistant donor 14SQE150025 (backcrossed from a Cucurbita ecuadorensis line, and then backcrossed with a derived Cucurbita pepo line) and a green zucchini (15SQE310133) lacking resistance factors to potivirus were genotyped using 190 genetic markers across the entire genome, and gene maps were calculated. Polymorphic markers were identified and selected to distinguish parents with the smallest set of analytical markers on their genome, based on the allele patterns and locations of a set of 3040 SNP markers. Each F2 plant was individually self-pollinated, and 173 F3 families isolated for PRSV resistance factors were obtained in sufficient seed quantity and quality for further phenotypic analysis. These F3 family plants were grown and evaluated for papaya ring spot virus resistance as described in Examples 2A-C above.

[0159] QTL detection was performed using the R / qtl package in the R statistical framework. First, the genotype probabilities were calculated (in 1 cM increments) using the function "calc.genoprob". Haley-Knott regression was performed to obtain an approximation of the standard interval mapping results. Next, the function "stepwiseqtl", which provides a fully automated model selection forward / backward algorithm, was implemented. The LOD threshold for the main effect was determined by 10,000 reorders. This algorithm considers different possible interactions (e.g., epistasis). The QTL locations for multiple QTL models were narrowed down (maximum likelihood estimates) using the function "refineqtl". The defined QTL models were fitted using the function "fitqtl" to obtain estimates of the QTL effects.

[0160] Example 4: Identification of a single QTL associated with high PRSV resistance One QTL was identified based on the PRSV resistance phenotype from the F3 population. Table 2 shows the chromosomal location based on the public map Cp4.1 (Montero-Pau et al., 2018), the effect of the QTL as measured by the LOD score, and the percentage of variation in PRSV resistance explained by the QTL on chromosome 10. The QTL showed a semi-dominant effect in the F2-F3 population. The QTL associated with high PRSV resistance was identified in the F3 population family on chromosome 10 with an LOD score of 62.1. Figure 2 shows examples of plants that show PRSV sensitivity when QTL10 is absent compared to plants that show PRSV resistance when QTL10 is present.

[0161] [Table 2]

[0162] Example 5: Gene transfer of PRSV resistance-constituting sequence into a commercial background As part of the development of resistant pumpkin varieties, the conversion of several key parent lines in the breeding program was initiated by backcrossing. Subsequently, marker-assisted selection was performed on cultivated pumpkin lines from various genetic backgrounds and germplasm groups according to the identified main QTLs. After sufficient backcrossing (BC4-BC5) to transfer PRSV resistance into these lines and restore the desired agrochemical and horticultural traits, self-offspring plants were formed to result in backcross family homozygosity at the QTL locus. The converted breeding lines and their corresponding hybrids were screened and compared for papaya earring spot virus resistance in tests conducted in a growth chamber according to the phenotypic evaluations described in Figure 1 and Example 2 to verify the effect of the QTLs and the improvement in resistance. In parallel, phenotypic evaluation by disease screening was also used at various backcross levels during the conversion process to track the transfer of resistance genes and confirm the efficiency of the markers in the early stages. Gene sequences associated with high resistance to PRSV derived from Cucurbita ecuadorensis were introduced into various lines of green zucchini by segregating backcross families and evaluating resistance levels in the BC1F2, BC2F2, and BC3F2 generations after the artificial testing described in Example 2.

[0163] The gene-transferred lines emphasized specific phenotypes similar to the recurrent parent phenotype in terms of the characteristics of open, upright plants with green cylindrical or teardrop-shaped bodies, while also containing gene transfer sequences favorable for high PRSV resistance. The results of phenotypic testing, along with the results of testing for the presence or absence of representative markers in QTL10, are summarized in Table 3 below for various genetic backgrounds. The converted lines were evaluated for PRSV resistance under control conditions, and the improvement in resistance levels to PRSV was observed by comparing them with the recurrent parent lines.

[0164] We retained plant 22SQE800260, a descendant resulting from a cross between a resistant and susceptible line, and deposited it with NCIMB under NCIMB accession number 44148. Pumpkin plant 22SQE800260 is heterozygous for the PRSV resistance trait; that is, it contains one copy of the gene transfer sequence that confers papayaring spot virus resistance from the Cucurbita ecuadorensis line, and also possesses the ZYMV resistance QTL Zn in a homozygous state.

[0165] [Table 3]

[0166] Various recurrent parents of the green zucchini pumpkin species (plants 2, 4, and 6) exhibit a range of moderate to severe susceptibility phenotypes. Differences in the levels of resistance to PRSV among the original recurrent parents may be related to the presence of additional resistance factors to other pottyviruses (ZYMV, WMV). Converted lines containing SNP markers spanning the underlying gene transfer sequence of QTL10 showed increased PRSV resistance compared to their unconverted repeats and achieved disease scores higher than 8.

[0167] In this region, twelve SNP markers within the QTL interval—AXQ15932, AXQ10805, AXQ16147, AXQ16199, AXQ6853, AXQ18924, AXQ10561, AXQ18164, AXQ1410, AXQ12317, AXQ14001, and AXQ9337—show specificity in the selection of donor resistance alleles derived from resistant donors, and among these, the SNP marker AX16199 is most closely associated with resistance.

[0168] Table 4 shows both the genetic and physical locations of the QTL on chromosome 10, as well as the locations of 12 SNP markers closely associated with the QTL. The physical locations are provided by referring to the publicly available genome assembly of Cp4.1LG10 (Montero-Pau et al., 2018).

[0169] [Table 4]

[0170] Example 6: Sequence and SNP marker information for QTL10 The sequence information for Axiom markers 1-12 (AXQ15932, AXQ10805, AXQ16147, AXQ16199, AXQ6853, AXQ18924, AXQ10561, AXQ18164, AXQ1410, AXQ12317, AXQ14001, AXQ9337) is summarized in Table 5 below.

[0171] [Table 5]

[0172] For example, SNP marker 1 (AXQ1593) at position 1985257 bp on chromosome 10 (based on the publicly available genome assembly Cp4.1) is characterized by a specific sequence polymorphism (resistance-donor allele versus susceptibility-allele) at position 36 of the target sequence of Sequence ID No. 1. Sequence ID No. 1 provides the target resistance sequence.

[0173] Example 7: Sequence and SNP marker information for ZYMV-resistant QTL allele Zn Furthermore, lines possessing additional potivirus resistance, such as the ZYMV-resistant QTL allele Zn, were found to exhibit higher resistance levels to RSV compared to lines converted solely with PRSV-highly resistant QTL10 lacking any additional potivirus resistance. These results are summarized in Table 6 and Figure 3 below.

[0174] [Table 6]

[0175] The gene map information for the Zn allele, as well as the sequence information for the specific SNP marker SQ0005B and its corresponding PCR primer / probe, are summarized in Tables 7 and 8 below. This marker can be used to detect the ZYMV-resistant Zn allele when the gene is transferred into a Cucurbita pepo background. This can be done using deposit material 22SQE800260 or other existing publicly available sources such as the Cucurbita pepo cultivar 268NiW deposited under NCIMB accession number 41727.

[0176] [Table 7]

[0177] [Table 8]

[0178] References ·Food and Agriculture Organization of the United Nations, Statistics Division, FAOSTAT. http: / / www.fao.org / faostat / en / #home ·Frost,2015,Identifying and marketing quality open-pollinated and organic cucurbit seedstocks for Virginia,Final report for FS13-273. https: / / projects.sare.org / project-reports / fs13-273 / ·Maluf et al.,1986,Screening of Cucurbita spp.Accessions for resistance to Watermelon MosaicVirus-1.Braz J Genet. 9:161-167. ·Montero-Pau et al.,2018,De novo assembly of the zucchini genome reveals a whole-genome duplication associated with the origin of the Cucurbita genus,Plant Biotechnol.J.16(6),p1161-1171. ·Omar et al.,2011,Egyptian isolates of Papaya ringspot virus form a molecularly distinct clade.Journal of Plant Pathology. ·Provvidenti et al.,1978,Resistance in feral species to six viruses infecting cucurbita.Plant Disease Report 62:326-329. ·United States Department of Agriculture,National Agricultural Statistics Service,Vegetables 2019 Summary. https: / / www.nass.usda.gov / Publications / Todays_Reports / reports / vegean20.pdf ·Ward and Shukla,1991,Intervirology 32(5):269-96.doi:10.1159 / 000150211.

Claims

1. A cultivated pumpkin plant resistant to papaya ringspot virus (PRSV) infection, preferably a cultivated Cucurbita pepo plant, wherein its genome contains a gene transfer sequence derived from Cucurbita ecuadolensis that confers resistance to PRSV, the gene transfer sequence located on chromosome 10, and the following SNP markers: a) A genotype that is heterozygous or homozygous for SNP marker 1 at the position corresponding to position 36 of Sequence ID No. 1; b) G genotypes that are heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) G genotypes that are heterozygous or homozygous for SNP marker 6 at position 36 of sequence number 6; g) C genotypes that are heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) C genotypes that are heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for the SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for the SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or l) Genotype A that is heterozygous or homozygous for the SNP marker 12 at the position corresponding to position 36 of sequence number 12. A plant containing at least one of the following.

2. The plant according to claim 1, wherein the gene transfer sequence includes at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and / or SEQ ID NO: 12, or a sequence that is at least 80% identical to one or more of the aforementioned sequences.

3. The plant according to claim 1 or 2, wherein the plant is homozygous for at least one SNP marker.

4. The plant according to any one of claims 1 to 3, further comprising a Zn allele located on chromosome 16 and associated with resistance to zucchini yellow mosaic virus (ZYMV), wherein the Zn allele includes a G genotype that is homozygous for the SNP marker 13 at position 119 of sequence number 13.

5. The plant according to any one of claims 1 to 4, wherein the gene transfer sequence is contained in the Cucurbita pepo plant 22SQE800260 (its representative seeds are deposited under NCIMB accession number 44148), or its offspring or ancestor.

6. A plant according to any one of claims 1 to 5, obtained by crossing a Cucurbita pepo plant 22SQE800260 (its representative seeds are deposited under NCIMB accession number 44148), or its offspring or ancestor, with a pumpkin plant that does not contain the gene transfer sequence that confers PRSV resistance.

7. A plant body according to any one of claims 1 to 6, which is an inbred line, a diploid haploid, a diploid, or a hybrid plant body.

8. The plant portion of a plant body according to any one of claims 1 to 7.

9. Seeds that produce a plant body or plant part according to any one of claims 1 to 8.

10. A method for producing PRSV-resistant cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, a) A step of crossing a plant according to any one of claims 1 to 7 with a cultivated pumpkin plant lacking the gene transfer sequence that confers PRSV resistance, b) A step of selecting a progeny plant containing the gene transfer sequence located on chromosome 10 that confers resistance to PRSV, the following SNP marker: i) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of sequence number 1; ii) G genotypes that are heterozygous or homozygous for SNP marker 2 at position 36 of sequence number 2; iii) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; iv) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; v) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; vi) G genotypes that are heterozygous or homozygous for SNP marker 6 at position 36 of sequence number 6; vii) C genotypes that are heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; viiii) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; ix) C genotype that is heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; x) A G genotype that is heterozygous or homozygous for the SNP marker 10 at the position corresponding to position 36 of sequence number 10; xi) A genotype that is heterozygous or homozygous for the SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or xi) A genotype that is heterozygous or homozygous for the SNP marker 12 at the position corresponding to position 36 of sequence number 12. A selection step which includes detecting at least one of the following A method for producing plants that contain, thereby enhancing resistance to PRSV.

11. c) Propagating the selected offspring, or crossbreeding the selected offspring with another pumpkin plant to produce further offspring. The method according to claim 10, further comprising:

12. The method according to claim 11, further comprising selecting offspring and allowing them to self-pollinate / cross-breed for a further 2 to 10 generations.

13. The plant according to claim 10, further comprising the step of detecting a G genotype that is homozygous for the SNP marker 13 at the position corresponding to position 119 of sequence number 13.

14. The method according to any one of claims 10 to 13, wherein the plant in step a) is a Cucurbita pepo plant 22SQE800260 (its representative seeds are deposited under NCIMB accession number 44148) or its descendants or ancestors.

15. A method for producing F1 pumpkin plants resistant to PRSV, comprising crossing an inbred pumpkin plant, which is a plant described in any one of claims 1 to 7, with a different inbred pumpkin plant to produce F1 hybrid offspring.

16. A method for identifying cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, that exhibit resistance to PRSV infection and possess at least one copy of a gene transfer sequence that confers PRSV resistance, wherein the following SNP markers are used: a) A genotype that is heterozygous or homozygous for SNP marker 1 at the position corresponding to position 36 of Sequence ID No. 1; b) G genotypes that are heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) G genotypes that are heterozygous or homozygous for SNP marker 6 at position 36 of sequence number 6; g) C genotypes that are heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) C genotypes that are heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for the SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for the SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or l) Genotype A that is heterozygous or homozygous for the SNP marker 12 at the position corresponding to position 36 of sequence number 12. A method comprising the step of detecting at least one of the following, thereby identifying a pumpkin plant that exhibits resistance to PRSV.

17. The method according to claim 16, further comprising selecting a pumpkin plant containing one or more of the SNP markers, and crossing the selected pumpkin plant with a second pumpkin plant to produce offspring pumpkin plants containing at least one of the SNP markers and exhibiting resistance to PRSV.

18. A method for producing pumpkin seeds, comprising growing a pumpkin plant from the seeds described in claim 9, and causing the plant to produce further pumpkin seeds.

19. A method for evaluating the genotype of cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, that exhibit resistance to PRSV, a) A step of preparing a sample from the plant body, b) A step of detecting a QTL locus located on chromosome 10 and associated with PRSV resistance in the sample, wherein the QTL locus is identified by the following SNP marker: i) A genotype that is heterozygous or homozygous for SNP marker 1 at position 36 of sequence number 1; ii) G genotypes that are heterozygous or homozygous for SNP marker 2 at position 36 of sequence number 2; iii) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; iv) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; v) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; vi) G genotypes that are heterozygous or homozygous for SNP marker 6 at position 36 of sequence number 6; vii) C genotypes that are heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; viiii) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; ix) C genotype that is heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; x) A G genotype that is heterozygous or homozygous for the SNP marker 10 at the position corresponding to position 36 of sequence number 10; xi) A genotype that is heterozygous or homozygous for the SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or xi) A genotype that is heterozygous or homozygous for the SNP marker 12 at the position corresponding to position 36 of sequence number 12; and / or xiiii) Any other DNA marker associated with the QTL locus, sandwiched between SNP markers 1 and 12 A step including at least one of the following A method that includes this.

20. A method for identifying gene transfer sequences associated with high resistance to PRSV in cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, comprising the step of detecting an allele of at least one DNA marker genetically associated with the QTL locus associated with the high resistance to PRSV in the plant, wherein the allele is located within 10 cM, preferably within 5 cM, of the QTL locus located on chromosome 10 in a genomic region flanked by SNP markers 1 and 12.

21. The aforementioned QTL locus is identified by the following SNP markers. a) A genotype that is heterozygous or homozygous for SNP marker 1 at the position corresponding to position 36 of Sequence ID No. 1; b) G genotypes that are heterozygous or homozygous for SNP marker 2 at the position corresponding to position 36 of Sequence ID No. 2; c) A T genotype that is heterozygous or homozygous for SNP marker 3 at the position corresponding to position 36 of sequence number 3; d) A G genotype that is heterozygous or homozygous for SNP marker 4 at the position corresponding to position 36 of sequence number 4; e) A G genotype that is heterozygous or homozygous for SNP marker 5 at the position corresponding to position 36 of sequence number 5; f) G genotypes that are heterozygous or homozygous for SNP marker 6 at position 36 of sequence number 6; g) C genotypes that are heterozygous or homozygous for SNP marker 7 at the position corresponding to position 36 of sequence number 7; h) A genotype that is heterozygous or homozygous for SNP marker 8 at position 36 of sequence number 8; i) C genotypes that are heterozygous or homozygous for SNP marker 9 at position 36 of sequence number 9; j) A G genotype that is heterozygous or homozygous for the SNP marker 10 at the position corresponding to position 36 of sequence number 10; k) A genotype that is heterozygous or homozygous for the SNP marker 11 at the position corresponding to position 36 of sequence number 11; and / or l) Genotype A that is heterozygous or homozygous for the SNP marker 12 at the position corresponding to position 36 of sequence number 12. The method according to claim 20, which can be specified by at least one of the following.

22. The method according to claim 21, further comprising the step of selecting a cultivated pumpkin plant containing the gene transfer sequence, preferably a cultivated Cucurbita pepo plant.

23. A method for identifying cultivated pumpkin plants, preferably cultivated Cucurbita pepo plants, that exhibit high resistance to PRSV by identifying QTLs associated with high resistance to PRSV, a) A step of detecting at least one DNA marker derived from a pumpkin plant (this DNA marker is associated with a chromosomal segment associated with high resistance to PRSV), wherein the chromosomal segment is flanked on both sides by SNP markers having at least 80% sequence identity to SEQ ID NOs: 1 and 12; b) The step of identifying the pumpkin plant containing the at least one DNA marker A method that includes this.

24. A method for identifying a wild pumpkin source with PRSV resistance trait on chromosome 10, a) To provide one or more wild pumpkin varieties; b) Screening one or more wild pumpkin strains using a kit that detects at least one of the SNP markers listed in Table 5, c) Identify a wild pumpkin strain containing at least one of the SNP markers selected from the list in Table 5. A method that includes this.

25. Use of any of Sequence IDs 1-12 to screen a population of pumpkin plants for the presence of a QTL locus located on chromosome 10 that is associated with high PRSV resistance.

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