durable resistance of spinach to downy mildew

Abstract

The present invention relates to methods for identifying and selecting plants resistant to downy mildew and other plant pathogens. Methods for identifying new genes encoding proteins associated with plant resistance to downy mildew and other plant pathogens and uses thereof are provided. The disease resistance genes are useful for producing resistant plants through breeding, transgenic modification, or genome editing.

Description

[0001] sequence list

[0002] This application contains a sequence list submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on July 25, 2022, named "20220725 PVS-023-EP-WO Sequencelisting ST.26.xml" and is 1084 kilobytes in size. Technical Field

[0003] This invention relates to the fields of plant breeding and molecular biology, and more specifically to novel genes that provide resistance to downy mildew and their applications. Background Technology

[0004] Spinach (Spinacia oleracea) is a flowering plant belonging to the Amaranthaceae family, cultivated as a vegetable. The edible part of spinach is its leaves during its vegetative stage. Spinach is sold in bulk, bundled, in pre-packaged bags, canned, or frozen. There are three basic types of spinach: curly-leaf, semi-curly-leaf, and flat-leaf. Curly-leaf spinach has wrinkled, curled leaves. Flat-leaf or smooth-leaf spinach has generally broad, smooth leaves. Semi-curly-leaf spinach is a variety with slightly curled leaves. The main market for spinach is its tender leaves. Tender spinach leaves are usually from the flat-leaf variety, and the harvested leaves are typically no longer than about 8 cm. These soft, sweet leaves are sold in bulk rather than in bundles. They are often used in salads but can also be lightly cooked. Spinach downy mildew is caused by the pathogen *Peronospora effusa* (also known as spinach powdery mildew, P. farinosaf.sp.spinaciae), and is a major threat to spinach growers because it affects the harvested part of the plant, namely the leaves. Infection renders the leaves unsuitable for sale and consumption, phenotypically manifesting as yellow spots on older leaves and the observation of light gray fungal growth on the surface of abaxial leaves. Infection can spread very rapidly and can occur in greenhouse cultivation, vertical farming, and soil cultivation. The optimal temperature for *Peronospora effusa* formation and germination is 9–12°C, and high relative humidity promotes this process. When the pathogen is deposited on moist leaf surfaces, it easily germinates and infects the leaves. The pathogen grows best at 8–20°C and ≥80% relative humidity, and growth can be observed within 6–13 days after infection. *Peronospora effusa* can survive in soil for up to 3 years, or in seeds or living plants. Various plants or germplasm known to possess at least partial resistance to downy mildew caused by *Spinacia oleracea*, such as those from Monsanto Vegetable Intellectual Property Management (Spinacia oleracea L., Kona-Swb2636, and Smbsl51262-SmbS015-1262m), She Hongbing et al. 2018 (Fine mapping and candidate gene screening of the downy mildewresistance gene RPFl in Spinach), and WO2013 / 064436. Methods for regulating plant growth and phenotype are known in this art, for example, from US 2017 / 037422. Furthermore, various spinach genes are known and available from the NCBI database. In recent years, various resistance genes that provide resistance to downy mildew in spinach plants have been identified. However, it has been observed that existing resistant spinach cultivars may become susceptible to the pathogen again. Studies have shown that the cultivars themselves have not changed, therefore the loss of downy mildew resistance must be due to *Spinacia oleracea* overcoming the resistance of these spinach cultivars.Downy mildew races capable of infecting resistant spinach cultivars have been identified in a differentiation reference set used to test resistance in spinach cultivars. The differentiation set contains a range of spinach cultivars (hybrids) with different resistance patterns to the currently identified pathogenic races. Currently, 19 pathogenic races (Pe) of spinach downy mildew have been formally identified and described. Between 1990 and 2009, races 4 to 10 were identified, demonstrating the diversity and adaptability of the pathogen in overcoming spinach resistance. In 2014, the Correll laboratory at the University of Arkansas identified the isolate UA1014APLP (also known as UA1014, now Pe:17). The International Working Group on Spinach Downy Mildew (IWGP) has named two new spinach spreading downy mildew races Pe:18 and Pe:19. These two races pose a significant threat to the spinach industry.

[0005] Different combinations of pathogenic races or isolates can occur in different geographical regions. Therefore, the spinach industry has a strong need for spinach cultivars that are resistant to as many relevant downy mildew races as possible, preferably resistant to all races that may appear in the region, and even to the latest threats that cannot be countered by the resistance present in commercially available spinach cultivars.

[0006] It is crucial to remain at the forefront of this field, as the ability of downy mildew to disrupt resistances present in commercially available spinach cultivars continues to evolve. Therefore, novel resistance genes are invaluable assets and constitute a significant research focus in spinach breeding. Given the threat of not being able to find new R genes against emerging pathogen races, there is also a need to search for broad-spectrum (most / all races) and more durable alternative resistances.

[0007] Spinach breeders aim to rapidly develop spinach varieties resistant to as many *Pe* races as possible, including those recently identified. Currently, 19 *Pe* races are officially recognized and available to the public from the Department of Plant Pathology at the University of Arkansas (Fairytville, AR 72701, USA) and from NAK Tuinbouw, Sotaweg 22, 2371 GD Roelofarendsveen, Netherlands. *Pe.20* has been identified in some regions, and further *Pe* races are expected to continue to emerge. Summary of the Invention

[0008] This document provides compositions and methods for identifying and selecting plant disease susceptibility genes, or "S genes." These compositions and methods can be used to produce resistant plants through chemical mutagenesis, selection of disease-resistant plants, creation of transgenic resistant plants, and / or creation of resistant genome-edited plants. This document also provides plants exhibiting newly acquired or enhanced resistance to various plant diseases compared to control plants. In some embodiments, the compositions and methods can be used to produce resistant plants through chemical mutagenesis, selection of spinach plants resistant to downy mildew (DM), creation of transgenic DM-resistant spinach plants, and / or creation of DM-resistant genome-edited spinach plants.

[0009] DM-resistant spinach plants can be crossed with a second spinach plant to obtain progeny spinach plants carrying the resistance gene allele. Disease resistance can be newly acquired or enhanced compared to control plants lacking the favorable allele. The DM resistance gene allele can be further refined to be identified by markers and include chromosomal regions that define the markers. In some embodiments, methods for identifying and / or selecting DM-resistant plants are shown. In these methods, the presence of resistance gene alleles on chromosome 4, associated with DM resistance, is analyzed in the DNA of spinach plants. Specifically, the resistance gene alleles contain "T" in Spov3_chr4_93275081 (reference sequence SEQ ID NO: 241, position 101), "A" in Spov3_chr4_105049870 (reference sequence SEQ ID NO: 242, position 101), "A" in Spov3_chr4_107241402 (reference sequence SEQ ID NO: 243, position 101), "A" in Spov3_chr4_109546568 (reference sequence SEQ ID NO: 244, position 101), and "A" in Spov3_chr4_109698808 (reference sequence SEQ ID NO: 243, position 101). The following sequences contain the character "A": SEQ ID NO: 245 (101st bit); Spov3_chr4_112008390 (SEQ ID NO: 246 (101st bit)); Spov3_chr4_112574123 (SEQ ID NO: 247 (101st bit)); Spov3_chr4_117783935 (SEQ ID NO: 248 (101st bit)); Spov3_chr4_118191085 (SEQ ID NO: 249 (101st bit)); Spov3_chr4_118788268 (SEQ ID NO: 250 (101st bit)); or Spov3_chr4_121142541 (SEQ ID NO: 247 (101st bit)). NO: 251 (position 101) contains “A”, and if the resistance gene allele is detected, the plant is identified and / or selected as having DM resistance.

[0010] In some embodiments, methods for identifying and / or selecting plants resistant to DM include detecting or selecting genomic segments comprising SEQ ID NO: 1, SEQ ID NO: 93, or SEQ ID NO: 169. DM resistance may be newly conferred or enhanced relative to control plants lacking favorable alleles. In one embodiment, the DM-resistant segment comprises a gene encoding a subunit protein of a clathrin aptamer that conferred or enhanced resistance to DM. In some embodiments, the subunit protein of the clathrin aptamer comprises an amino acid sequence as shown in SEQ ID NO: 3. In one embodiment, the DM-resistant segment comprises a gene encoding a pentatricopeptiderepeat-containing protein that conferred or enhanced resistance to DM. In some embodiments, the pentatricopeptiderepeat-containing protein comprises an amino acid sequence as shown in SEQ ID NO: 95. In one embodiment, the DM-resistant segment comprises a gene encoding an RPS2-like resistance protein that conferred or enhanced resistance to DM. In some embodiments, the RPS2-like resistance protein comprises an amino acid sequence as shown in SEQ ID NO: 171.

[0011] In another embodiment, a method is provided for identifying and / or selecting plants resistant to DM, wherein one or more marker alleles linked to and associated with any of the following are detected in the plant: “T” at Spov3_chr4_93275081 (reference sequence SEQ ID NO: 241, position 101), “A” at Spov3_chr4_105049870 (reference sequence SEQ ID NO: 242, position 101), “A” at Spov3_chr4_107241402 (reference sequence SEQ ID NO: 243, position 101), “A” at Spov3_chr4_109546568 (reference sequence SEQ ID NO: 244, position 101), and Spov3_chr4_109698808 (reference sequence SEQ ID NO: 243, position 101). The following are examples of string positions: "A" at position 101 of SEQ ID NO: 245, "A" at position 101 of Spov3_chr4_112008390 (SEQ ID NO: 246), "T" at position 101 of Spov3_chr4_112574123 (SEQ ID NO: 247), "T" at position 101 of Spov3_chr4_117783935 (SEQ ID NO: 248), "A" at position 101 of Spov3_chr4_118191085 (SEQ ID NO: 249), "A" at position 101 of Spov3_chr4_118788268 (SEQ ID NO: 250), or "A" at position 101 of Spov3_chr4_121142541 (SEQ ID NO: 245). The "A" at position 101 (NO: 251) was selected, and plants with more than one marker allele were chosen. More than one marker allele could be linked on a single meiotic genetic map at map distances of less than 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, or 0.1 cM.The selected plants can be crossed with a second plant to obtain progeny plants having one or more marker alleles linked to and associated with any of the following: “T” at Spov3_chr4_93275081 (reference sequence SEQ ID NO: 241, position 101), “A” at Spov3_chr4_105049870 (reference sequence SEQ ID NO: 242, position 101), “A” at Spov3_chr4_107241402 (reference sequence SEQ ID NO: 243, position 101), “A” at Spov3_chr4_109546568 (reference sequence SEQ ID NO: 244, position 101), “A” at Spov3_chr4_109698808 (reference sequence SEQ ID NO: 245, position 101), and Spov3_chr4_112008390 (reference sequence SEQ ID NO: 245, position 101). The following are examples of sequences with different positions: "A" at position 101 of SEQ ID NO: 246, "T" at position 101 of SEQ ID NO: 247 (reference sequence), "T" at position 101 of SEQ ID NO: 248 (reference sequence), "A" at position 101 of SEQ ID NO: 249 (reference sequence), "A" at position 101 of SEQ ID NO: 250 (reference sequence), or "A" at position 101 of SEQ ID NO: 251 (reference sequence).

[0012] In another embodiment, a method for introgression of gene alleles associated with DM resistance is shown herein. In these methods, a plant population is screened with more than one marker to determine whether any plant possesses a gene allele associated with DM resistance, and at least one plant possessing a gene allele associated with DM resistance is selected from the population. The gene alleles contain “T” in Spov3_chr4_93275081 (reference sequence SEQ ID NO: 241, position 101), “A” in Spov3_chr4_105049870 (reference sequence SEQ ID NO: 242, position 101), “A” in Spov3_chr4_107241402 (reference sequence SEQ ID NO: 243, position 101), “A” in Spov3_chr4_109546568 (reference sequence SEQ ID NO: 244, position 101), “A” in Spov3_chr4_109698808 (reference sequence SEQ ID NO: 245, position 101), and “A” in Spov3_chr4_112008390 (reference sequence SEQ ID NO: 241, position 101). The following sequences contain the character “A”: SEQ ID NO: 246 (101st bit); “T”: Spov3_chr4_112574123 (SEQ ID NO: 247 (101st bit); “T”: Spov3_chr4_117783935 (SEQ ID NO: 248 (101st bit)); “A”: Spov3_chr4_118191085 (SEQ ID NO: 249 (101st bit)); “A”: Spov3_chr4_118788268 (SEQ ID NO: 250 (101st bit)); or “A”: Spov3_chr4_121142541 (SEQ ID NO: 251 (101st bit)).

[0013] In some implementations, the introduction of genes resistant to DM from resistant lines into susceptible lines can be achieved through marker-assisted trait introgression, transgenesis, or gene editing (including gene substitution or allele substitution).

[0014] The methods implemented by this invention relate to: methods for transforming host cells, including plant cells, including transforming host cells with a polynucleotide according to one embodiment of the invention; methods for generating plants, including transforming plant cells with a recombinant DNA construct according to one embodiment of the invention and regenerating plants from the transformed plant cells; and methods for conferring or enhancing disease resistance, including transforming plants with the recombinant DNA construct disclosed herein.

[0015] In one embodiment, a method is provided to alter the expression level of a protein capable of conferring disease resistance in a plant or plant cell, wherein the method comprises transforming plant cells with a recombinant DNA construct disclosed herein and culturing the transformed plant cells under conditions suitable for expression of the recombinant DNA construct, wherein expression of the recombinant DNA construct results in an alteration in the expression level of a protein capable of conferring disease resistance in the transformed host.

[0016] This paper provides a method for altering plant disease resistance, the method comprising introducing one or more nucleotide modifications by targeting a DNA break at a locus in a plant, wherein the locus contains an S gene involved in disease resistance encoding a subunit protein of a clathrin aptamer, a family protein containing a triangular pentapeptide repeat sequence, or an RPS2-like resistance protein, and wherein plant disease resistance is altered compared to control plants without the more than one introduced genetic modification. In some embodiments, the S gene comprises a polynucleotide encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 4, 7-34, 96, 99-121, 172, 175-196, 252-270, 316-320, and 331, or encoding a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence selected from SEQ ID NOs: 6, 64-92, 98, 145-168, 174, 219-240, 294-315, 326-330, and 333. In some embodiments, the targeted DNA modification targets more than one different locus involved in plant disease resistance.

[0017] In some embodiments, the targeted DNA modification is selected from the group consisting of insertions, deletions, single nucleotide polymorphisms (SNPs), and polynucleotide modifications, thereby altering (increasing or decreasing) the expression of the polypeptide encoded by the S gene. In some embodiments, the targeted DNA modification results in one or more of the following: changes in S gene expression (increases or decreases); the production of one or more alternatively spliced ​​transcripts of the S gene; changes such as deletion of one or more DNA-binding domains; frameshift mutations in one or more exons of the S gene; changes such as deletion of a large portion of the S gene or deletion of the full-length open reading frame of the S gene; induction or repression of enhancer motifs present in the regulatory region encoding the S gene; modification of one or more nucleotides; or changes such as deletion of regulatory elements operatively linked to S gene expression, wherein the regulatory elements are present in a promoter, intron, 3'UTR, terminator, or a combination thereof. In some implementations, the targeted DNA modification targets the locus of the S gene such that one or more nucleotide modifications are present in (a) the same coding region; (b) the non-coding region; (c) the regulatory sequence; (d) the untranslated region of an endogenous polynucleotide encoding a polypeptide involved in disease resistance and significantly improving compatibility; or (e) any combination of (a)-(d).

[0018] In some implementations, targeted DNA modification is introduced via RNA-guided endonucleases, site-specific deaminases, or site-specific endonucleases. Targeted DNA modification is performed using genome modification techniques selected from the group consisting of polynucleotide-guided endonucleases, CRISPR-Cas endonucleases, base-editing deaminases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), engineered site-specific broad-spectrum nucleases, or Argonaute. In some embodiments, the targeted DNA modification is introduced using a guide RNA corresponding to a target sequence, the target sequence comprising a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 4, 7-34, 96, 99-121, 172, 175-196, 252-270, 316-320, and 331, or encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 6, 64-92, 98, 145-168, 174, 219-240, 294-315, 326-330, and 333. In some embodiments, when targeted DNA modification leads to alterations in the expression or activity of the protein encoded by the S gene, the plant exhibits enhanced disease resistance. In some embodiments, the plant is a spinach plant. In some embodiments, the disease is downy mildew.

[0019] This article also provides plants exhibiting enhanced disease resistance containing a modified locus of an S gene involved in disease resistance, wherein, compared to control plants, the locus contains one or more introduced mutations, and wherein the locus contains a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 4, 7-34, 96, 99-121, 172, 175-196, 252-270, 316-320, and 331, or encoding a polynucleotide containing a sequence selected from SEQ ID NOs: 4, 7-34, 96, 99-121, 172, 175-196, 252-270, 316-320, and 331. The polynucleotide of a polypeptide having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. NOs: 6, 64-92, 98, 145-168, 174, 219-240, 294-315, 326-330, and 333.

[0020] This document also provides recombinant DNA constructs comprising a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 4, 7-34, 96, 99-121, 172, 175-196, 252-270, 316-320, and 331, or encoding a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 6, 64-92, 98, 145-168, 174, 219-240, 294-315, 326-330, and 333, said polynucleotide being operatively linked to at least one heterologous nucleic acid sequence.

[0021] Furthermore, this document provides plant cells comprising a recombinant DNA construct comprising a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 4, 7-34, 96, 99-121, 172, 175-196, 252-270, 316-320, and 331, or encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 6, 64-92, 98, 145-168, 174, 219-240, 294-315, 326-330, and 333, said polynucleotide being operatively linked to at least one heterologous nucleic acid sequence.

[0022] This document provides guide RNA sequences for plant cell loci, wherein the loci comprise a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 4, 7-34, 96, 99-121, 172, 175-196, 252-270, 316-320, and 331, or encoding a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 6, 64-92, 98, 145-168, 174, 219-240, 294-315, 326-330, and 333. In some embodiments, the guide RNA is present in a recombinant DNA construct.

[0023] While several embodiments have been disclosed, other embodiments of the invention will become apparent to those skilled in the art through the following detailed description of illustrative embodiments of the invention. Therefore, the drawings and detailed description are to be regarded as illustrative rather than restrictive in nature. Attached Figure Description

[0024] Figure 1 The diagram shows SNP alleles providing EMS transformation information as a percentage of total read coverage. The plot shows the percentage of Phytozome: Soleracea_575_Spov3 (X-axis: 0-122 Mb) mentioned in the examples at its respective positions on Chr4 (Y-axis: %R and %S).

[0025] Figure 2 GWAS analysis of 91 EMS-induced SNPs and susceptibility / resistance scores for Pe:16 are shown. Manhattan plots of SNP significance scores and their locations on chromosomes 1 through 6 of Phytozome:Soleracea_575_Spov3 (mentioned in the examples) are shown. Eleven SNPs located distal to Chr4 showed highly significant correlations.

[0026] Figure 3 GWAS analysis of B11-509-specific SNPs and susceptibility / resistance scores for Pe:16 are shown. A Manhattan plot of significance scores for SNPs within the 112-118 Mb region of Chr4 is shown. Three SNPs in the 0.62 Mb region surrounding Spov3_chr4.04649 (RPS2 sample) showed a clear correlation.

[0027] Figure 4 The sequence alignment of the reassembled contig_59494 mutant B11-509EMS22-20 with the coding sequence of the novel, high-quality Viroflay gene model SOV1g044450 is shown. Identical residues are indicated by "*", while different residues are indicated by spaces: intron 1 (13-290 bp), G>A (296), G>C (1751), - / T (1755), deletion (1756-1946), - / G (1947), and G>A (2120). T>C (1177) and T>A (12007) are synonymous mutations.

[0028] Figure 5 The sequence alignments of the wild-type Viroflay gene model SOV1g044450 protein with the orthologous sequences of the translated mutant B11-509EMS22-20 are shown. Identical residues are indicated by "*", while different residues are indicated by spaces: K490N, L493C, and G614R.

[0029] Figure 6 Differential expression analysis of the Soleracea_575_Spov3 gene model Chr4.04649 (RPS2-like) is presented. The relative expression levels of wild-type B11-509 and the mutant EMS22-20 at four time points are shown in untreated (M, simulated control) and Pe:16-infected (I) samples. Compared to wild-type B11-509 plants, the expression level in the mutant plants was upregulated at all time points in the simulated control treatment (uninfected plants) and at three time points in the Pe:16-infected plants, indicating that RPS2-like gene expression is generally increased in EMS22-20 plants regardless of the treatment (simulated control or Pe:16).

[0030] Figure 7 The infection process of DM isolate Pe:16 on leaves of B11-509 and EMS22-20 was shown using trypan blue staining and microscopic analysis. The infection process in B11-509 is illustrated, showing appressorium penetration of the leaf and clearly observable hyphal growth. Furthermore, haustoria formation is believed to occur in B11-509. Conversely, hyphal penetration was not observed in the mutant EMS22-20.

[0031] Figure 8 The image shows a microscopic analysis of the infection process of DM isolate Pe:16 in mutant EMS22-20 on days 4 and 6 (bottom right). It shows spores and hyphae accumulating together on the leaf surface and tending to elute from the leaf. Detailed Implementation

[0032] To facilitate a clearer understanding of the invention, certain terms are first defined. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Various methods and materials similar to, modified, or equivalent to those described and used herein can be used to implement embodiments of the invention without extensive experimentation; preferred materials and methods are described herein. In describing and claiming embodiments of the invention, the following terms will be used as defined below.

[0033] It will be understood that all terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limited in any manner or scope. For example, as used in this specification and the appended claims, the singular form may include plural references unless explicitly stated otherwise. Similarly, the word “or” is intended to include “and” unless the context explicitly states otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list. Furthermore, any units, prefixes, and symbols may be represented in a form recognized by the International System of Units (SI).

[0034] The numerical ranges given in this specification include the numbers within the defined range and include every integer within the defined range. Throughout this disclosure, various aspects of the invention are expressed in the form of ranges. It should be understood that the descriptions in range form are for convenience and brevity only and should not be construed as immutably limiting the scope of the invention. Therefore, the description of a range should be understood to include all possible subranges, fractions, and individual numerical values ​​specifically disclosed within that range. For example, a range description such as 1 to 6 should be understood to include specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, and decimals or fractions, such as 1.2, 3.8, 1, etc. 1 / 2 and 4 3 / 4. The above applies regardless of the width of the range.

[0035] As used herein, the term "about" refers to variations in quantity that may be caused by, for example, conventional measuring techniques and equipment, including but not limited to, mass, volume, time, and temperature. Furthermore, considering the solid and liquid handling procedures used in the real world, there are likely some unintentional errors and variations that may arise from differences in the manufacture, origin, or purity of the ingredients used to manufacture the composition or to carry out the methods, etc. The term "about" also includes these variations. Whether or not modified by the term "about," the claims include equivalents of quantity.

[0036] An allele is "associated" with a trait when it is part of or linked to a DNA sequence that influences phenotypic expression. The presence of an allele indicates how the trait will be expressed.

[0037] As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA located on a single chromosome in a plant. Genetic elements or genes located on a single chromosomal interval are physically linked. There is no particular limitation on the size of a chromosomal interval. In some respects, genetic elements located within a single chromosomal interval are genetically linked, and the genetic recombination distance is typically, for example, less than or equal to 20 cM or less than or equal to 10 cM. ​​That is, the frequency of recombination between two genetic elements within a single chromosomal interval is less than or equal to 20% or 10%.

[0038] In this application, the term "closely linked" means that the frequency of recombination between two linked sites is equal to or less than about 10% (i.e., they are no more than 10 cM apart on the genetic map). In other words, closely linked sites co-segregate at least 90% of the time. These are particularly useful to the subject matter of the invention when demonstrating a significant probability of co-segregation (linkage) between marker sites and desired traits (e.g., resistance to southern maize rust). The recombination frequency between closely linked sites, such as marker sites, and second sites can be less than 10%, preferably less than 9%, more preferably less than about 8%, more preferably less than about 7%, more preferably less than about 6%, more preferably less than about 5%, more preferably less than about 4%, more preferably less than about 3%, and more preferably less than about 2%. In a highly preferred embodiment, the associated sites exhibit recombination frequencies of less than about 1%, less than about 0.75%, more preferably less than about 0.5%, or more preferably less than about 0.25%. Two loci located on the same chromosome and whose recombination frequency between them is less than 10% (e.g., approximately 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25%) are also considered "proximate." In some cases, two different markers may have the same genetic map coordinates. In these cases, the two markers are so close that the recombination frequency between them is so low as to be undetectable.

[0039] The term "hybrid" or "hybrid" refers to sexual hybridization and involves the fusion of two haploid embryos to produce diploid offspring (e.g., cells, seeds, or plants). The term also includes one plant being pollinated by another plant or by itself (self-pollination, such as when pollen and ovules come from the same plant).

[0040] As used herein, "disease resistance" or "resistance to disease" refers to enhanced resistance to disease in plants compared to control plants. Disease resistance can manifest as fewer and / or smaller lesions, improved plant health, increased yield, increased root quality, increased plant vigor, less or no discoloration, improved growth, reduced necrotic area, or reduced wilting. In some embodiments, alleles can express resistance to more than one disease.

[0041] Diseases affecting spinach plants include, but are not limited to, anthracnose (Colletotrichum dematium f.sp.spinaciae), damping-off / seedling blight (Pythium ultimum), downy mildew (Peronospora effusa), wilt (Fusarium oxysporum f sp.spinaciae), creeping scab (Stemphylium versicarium, Stemphylium beticola, Stemphylium drummondii), yellow wilt (Verticillium dahlia), white rust (Albugo occidentalis), black root rot (Aphanomyces cochlioides), and cladosporium leaf spot (Cladosporium variabile). Therefore, the pathogens causing these diseases are widely distributed across multiple phyla. For example, species of the genus *Peronospora* are part of the phylum Oomycota, while species of the genus *Colletotrichum* are part of the phylum Ascomyceta.

[0042] Diseases affecting basil plants include, but are not limited to, downy mildew (Peronospora belbahrii).

[0043] Diseases affecting common legumes include, but are not limited to, anthracnose (Colletotrichumlindemuthianum), Pythium root rot (Pythium sclerotiorum), and white mold (Sclerotinia sclerotiorum).

[0044] Diseases affecting sugar beet plants include, but are not limited to, Pythium irregulare, damping-off / seedling blight, downy mildew, and Phytophthoradrechsleri.

[0045] Diseases affecting carrot plants include, but are not limited to, hollow spot (Pythium species), lateral root blight (Pythium species), leaf blight (Alternana dauci), powdery mildew (Erysiphe heradei), and white mold (Sclerotinia sclerotiorum).

[0046] Diseases affecting cucurbitaceous plants (such as cucumbers, melons, watermelons, squash, pumpkins, bottle gourds, and other crops) include, but are not limited to, Alternaria cucumerina leaf spot, Colletotrichum obiculare, Pseudoperonospora cubensis, Didymellabryoniae, and powdery mildew (Erysiphe cichoracearum and Sphaerothecafuliginea).

[0047] Diseases affecting grapevines include, but are not limited to, downy mildew (Plasmopara viticola).

[0048] Diseases affecting hop plants include, but are not limited to, downy mildew (Pseudoperonosporahumuli).

[0049] Diseases affecting lettuce plants include, but are not limited to, downy mildew (Bremia lactucae).

[0050] Diseases affecting Solanaceae plants (such as eggplant, pepper, petunia, potato, tomato, and tobacco) include, but are not limited to, powdery mildew (Leveillula taurica), wet rot (Phytophthora capsica), late blight (Phytophthora infestans), and penicillium (Peronosporahyoscyami f.sp.tabacina).

[0051] Diseases affecting rapeseed plants include, but are not limited to, black shank (Leptosphaeria maculan), damping-off (Rhizoctonia solani), and downy mildew (Peronospora parasitica).

[0052] Diseases affecting soybean plants include, but are not limited to, downy mildew (Peronospora manshurica).

[0053] Diseases affecting sunflower plants include, but are not limited to, downy mildew (Plasmopara halstedii).

[0054] Diseases affecting maize plants include, but are not limited to, brown downy mildew (Scleropthora rayssiae var. zeae), crazy top (Sclerophthora macrospora), downy mildew (Peronosclerospora sorghi), grass-root downy mildew (Sclerospora graminicola), Javanese downy mildew (Peronospora maydis), and Philippine downy mildew (Peronospora philippinensis).

[0055] Diseases affecting sorghum plants include, but are not limited to, downy mildew (specifically, Peronosclerosporasorghi in sorghum).

[0056] Compared to control plants, disease-resistant plants can exhibit increased resistance by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In some embodiments, compared to control plants, the plant health in the presence of the disease can be increased by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%.

[0057] "Backbone strains" are any strains bred and selected for superior agronomic performance.

[0058] As used in this article, the term “expression” generally refers to the production of a functional end product, such as mRNA or protein (precursor or mature).

[0059] A "favorable allele" is an allele located at a specific locus (marker, QTL, gene, etc.) that confers or contributes an agronomically desired phenotype, such as disease resistance, and allows for the identification of plants possessing that agronomically desired phenotype. A favorable allele for a marker is a marker allele that segregates along with the favorable phenotype.

[0060] As used herein, “gene” includes nucleic acid fragments that express functional molecules, such as, but not limited to, specific protein-coding sequences and regulatory elements, such as before (5' non-coding sequence) and after (3' non-coding sequence).

[0061] "Genetic markers" are nucleic acids that exhibit polymorphism within a population, and whose alleles are detectable and distinguishable by more than one analytical method, such as RFLP, AFLP, isoenzymes, SNPs, SSRs, etc. The term also refers to nucleic acid sequences complementary to the genome sequence, such as nucleic acid sequences used as probes. Markers corresponding to genetic polymorphism among members of a population can be detected using methods recognized in this technical field. These methods include, for example, PCR-based sequence-specific amplification methods, detection of restriction fragment length polymorphism (RFLP), detection of isoenzyme markers, detection of polynucleotide polymorphisms via allele-specific hybridization (ASH), detection of amplified variable sequences in plant genomes, detection of autonomous sequence duplication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Recognized methods also include the detection of expressed sequence tags and SSR markers from EST sequences, as well as randomly amplified polymorphic DNA (RAPD).

[0062] "Germium" refers to genetic material derived from an individual (e.g., a plant), a group of individuals (e.g., a plant strain, variety, or family), or a clone from a strain, variety, species, or culture, or more generally, the genetic material of all individuals within a species or several species (e.g., a maize germplasm collection or an Andean germplasm collection). Germium can be part of an organism or cell, or can be isolated from an organism or cell. Typically, germplasm provides genetic material with a specific molecular composition that provides the physical basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues that can grow into new plants, or plant parts such as leaves, stems, pollen, or cells that can be cultured into complete plants.

[0063] As used in this article, "plant locus" generally refers to the location on a plant's chromosome where genes (such as polynucleotides) involved in disease resistance are found.

[0064] The term "homologous" refers to nucleic acid sequences derived from a common ancestral gene through natural or artificial processes (e.g., members of the same gene family) and therefore typically exhibit sequence similarity. Generally, the identity of homologous nucleic acids is sufficient to allow one sequence or its complement to selectively hybridize with other sequences under selective hybridization conditions. The term "selective hybridization" involves hybridizing a nucleic acid sequence with a specific target nucleic acid sequence under stringent hybridization conditions to a detectably higher degree than hybridization with a non-target nucleic acid sequence (e.g., at least 2-fold higher than background) and substantially excluding non-target sequences. The sequence identity of selectively hybridized sequences is approximately at least 80%, typically at least 90%, and the sequence identity with each other can be 95%, 97%, 99%, or 100%. Nucleic acids exhibiting at least some degree of homology with a reference nucleic acid can be singular or identical to a reference nucleic acid or its complement.

[0065] The term “introduction” refers to the provision of nucleic acids (e.g., expression constructs) or proteins into a cell. Introduction includes the addition of nucleic acids to eukaryotic or prokaryotic cells, where the nucleic acids can be integrated into the cell’s genome, and includes the transient provision of nucleic acids or proteins to the cell. Introduction includes methods involving stable or transient transformation, as well as sexual hybridization. Therefore, in the context of inserting nucleic acid fragments (e.g., recombinant DNA constructs / expression constructs) into a cell, “introduction” means “transfection” or “transformation” or “transduction” and includes the addition of nucleic acid fragments to eukaryotic or prokaryotic cells, where the nucleic acids can be integrated into the cell’s genome (e.g., chromosomal, plasmid, plasmid, or mitochondrial DNA), transformed into autonomous replicons, or transiently expressed (e.g., transfected mRNA).

[0066] The term "introgression" refers to the transfer of a desired allele from one genetic background to another. For example, introgression of a desired allele at a specific locus can be achieved through sexual hybridization of two parents of the same species, where at least one parent carries the desired allele in its genome. Alternatively, allele transfer can occur, for example, through recombination between two donor genomes in fused protoplasts, where at least one donor protoplast carries the desired allele in its genome. The desired allele can be detected, for example, at QTLs, transgenes, etc., using markers associated with the phenotype. Offspring containing the desired allele can be repeatedly backcrossed with lines having the desired genetic background and the desired allele selected, thereby fixing the allele in the selected genetic background. When this process is repeated more than twice, the "introgression" process is often referred to as a "backcross."

[0067] "Introgression fragment," "introgression portion," or "introgression section" refers to a segment (or part or section) of chromosome introduced into another plant of the same or related species through hybridization or traditional breeding techniques such as backcrossing. In other words, the introgression is the result of a breeding method called "introgression" (e.g., backcrossing). It should be understood that the term "introgression fragment" does not include the entire chromosome, but only a portion of it. The infiltrated fragment can be large, for example, even half a chromosome, but is preferably small, for example, less than about 15 Mb, such as less than about 10 Mb, less than about 9 Mb, less than about 8 Mb, less than about 7 Mb, less than about 6 Mb, less than about 5 Mb, less than about 4 Mb, less than about 3 Mb, less than about 2 Mb, less than about 1 Mb (equal to 1,000,000 base pairs) or less than about 0.5 Mb (equal to 500,000 base pairs), for example, less than about 200,000 bp (equal to 200,000 base pairs), less than about 100,000 bp (100 kb), less than about 50,000 bp (50 kb), less than about 25,000 bp (25 kb).

[0068] A "strain" or "line" is a group of individuals that are typically inbred to some extent and are usually homozygous, and are homozygous at most loci (isogenetic or near-isogenetic). A "sub-strain" is a subset of offspring that is genetically distinct from other similar subsets of inbred offspring from the same ancestor.

[0069] As used herein, the term "linkage" describes the degree to which one marker locus is associated with another marker locus or some other locus. The linkage between a molecular marker and a locus influencing a phenotype is defined as a "probability" or "adjusted probability." Linkage can be expressed as a desired boundary or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when they are less than 50, 40, 30, 25, 20, or 15 map units (or cM) apart in a single meiotic map (based on a genetic map of a population that has undergone one round of meiosis, such as F2; ​​an IB M2 map consists of multiple meiotic divisions). In some aspects, it is advantageous to define a range for linkage classification, such as 10 to 20 cM, 10 to 30 cM, or 10 to 40 cM. ​​The stronger the linkage of a marker to a second locus, the better the marker indicates the second locus. Therefore, "closely linked sites," such as marker sites and second sites, exhibit a recombination frequency of less than 10%, preferably less than 9%, more preferably less than about 8%, more preferably less than about 7%, more preferably less than about 6%, more preferably less than about 5%, more preferably less than about 4%, more preferably less than about 3%, and more preferably less than about 2%. In a highly preferred embodiment, the recombination frequency of associated sites is less than about 1%, less than about 0.75%, more preferably less than about 0.5%, or more preferably less than about 0.25%. Two sites located on the same chromosome and located at a distance where the recombination frequency between the two sites is less than 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25%) are also referred to as "closely linked." Since one cM is the distance between two markers with a recombination frequency of 1%, any marker is closely linked (genetically and physically) to any other marker that is closely adjacent (e.g., at a distance of 10 cM or less). Two closely linked markers on the same chromosome can be located within 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, or 0.25 cM of each other.

[0070] A "site" is a location on a chromosome, such as a nucleotide, gene, sequence, or marker.

[0071] A “marker” is a method of locating a site on a genetic or physical map, or on another linkage between a marker and a trait locus (a site that influences a trait). The location of a marker detection can be determined by detecting polymorphic alleles and their genetic map, or by hybridization, sequence matching, or amplification of sequences on a physically mapped site. Markers can be DNA markers (detecting DNA polymorphisms), proteins (detecting variations at the level of encoded polypeptides), or simply inherited phenotypes. DNA markers can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., from spliced ​​RNA or cDNA). Depending on the DNA marker technology, a marker can consist of complementary primers flanking the locus and / or complementary probes that hybridize to the polymorphic allele at the locus. DNA markers or genetic markers can also be used to describe genes, DNA sequences, or nucleotides on the chromosome itself (rather than components used to detect genes or DNA sequences), and are commonly used when DNA markers are associated with specific traits in human genetics (e.g., markers for breast cancer). The term marker locus is the site (gene, sequence, or nucleotide) that the marker detects.

[0072] The marker can be determined by the type of polymorphism it detects and the marker technology used to detect the polymorphism. Marker types include, but are not limited to, detection of restriction fragment length polymorphism (RFLP), detection of isoenzyme markers, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), detection of simple sequence repeats (SSR), detection of amplified variable sequences in the plant genome, detection of autonomous sequence replication, or detection of single nucleotide polymorphisms (SNPs). SNPs can be detected by, for example, DNA sequencing, PCR-based sequence-specific amplification methods, detection of polynucleotide polymorphisms via allele-specific hybridization (ASH), dynamic allele-specific hybridization (DASH), molecular beacons, micromatrix hybridization, oligonucleotide ligand testing, Flap endonucleases, 5' endonucleases, primer extension, single-strand conformation polymorphism (SSCP), or temperature gradient gel electrophoresis (TGGE). DNA sequencing technologies such as pyrosequencing have the advantage of detecting a series of linked SNP alleles that make up a haplotype. Haplotypes often provide more information than SNPs (detecting higher levels of polymorphism).

[0073] "Marker allele" or "marker locus allele" can refer to one of several polymorphic nucleotide sequences found at the marker locus within a population.

[0074] Marker-assisted selection (MAS) is the process of selecting plant individuals based on marker genotypes. Marker-assisted anti-selection is the process of using marker genotypes to identify unselected plants for removal from breeding programs or planting.

[0075] A "marker locus" is a specific chromosomal location in the genome of a species where a particular marker can be found. Marker loci can be used to track the presence of second-linked sites, such as sites that influence the expression of phenotypic traits. For example, marker loci can be used to monitor the segregation of alleles at genetically or physically linked sites.

[0076] "Modified nucleotide" or "edited nucleotide" refers to a nucleotide sequence of concern that contains at least one change when compared to an unmodified nucleotide sequence. These "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0077] The term "molecular marker" can be used to refer to a genetic marker, as defined above, or its encoded product (e.g., a protein), used as a reference point in identifying linkage sites. Markers can originate from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from spliced ​​RNA, cDNA, etc.) or from encoded polypeptides. The term also refers to nucleic acid sequences complementary to or flanking the marker sequence, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A "molecular marker probe" is a nucleic acid sequence or molecule that can be used to identify the presence of a marker site, such as a nucleic acid probe complementary to the marker site sequence. Alternatively, in some respects, a marker probe refers to any type of probe capable of distinguishing (i.e., genotype) specific alleles present at the marker site. Nucleic acids are "complementary" when they hybridize specifically in solution. Some markers described herein are also called hybridization markers when located in insertion / deletion regions, such as the non-collinear regions described herein. This is because insertion regions are polymorphic by definition relative to plants without insertions. Therefore, the marker only needs to indicate the presence or absence of the insertion / deletion region. The hybridization markers can be identified using any appropriate marker detection technique, such as the SNP technique used in the examples provided herein.

[0078] The term "plant" includes the whole plant, plant cells, plant protoplasts, cultures of plant cells or tissues that can regenerate into a plant, plant callus, plant clumps, and complete plant cells in a part of a plant, such as seeds, flowers, cotyledons, leaves, stems, buds, roots, root tips, etc. As used herein, "modified plant" refers to any plant that has undergone genetic alterations due to human intervention. Modified plants can have genetic changes introduced through plant transformation, gene editing (including gene substitution or allele substitution), or conventional plant breeding.

[0079] The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,” “nucleic acid fragment,” and “isolated nucleic acid fragment” are used interchangeably herein. These terms include nucleotide sequences, etc. Polynucleotides can be polymers of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or modified nucleotide bases. Polynucleotides in the form of DNA polymers can contain fragments of more than one cDNA, genomic DNA, synthetic DNA, or mixtures thereof.

[0080] The terms “plasmid,” “vector,” and “cassette” refer to extrachromosomal elements that typically carry genes that are not part of the cell’s central metabolism and are usually in the form of circular double-stranded DNA fragments. These elements can be autonomously replicating sequences, genome-integrated sequences, bacteriophage or nucleotide sequences from any source, linear or circular, single-stranded or double-stranded DNA or RNA, wherein multiple nucleotide sequences are linked or recombined into a single construct capable of introducing promoter fragments and selected gene products, along with appropriate 3' untranslated sequences, into the cell.

[0081] As used herein, in the context of expression (e.g., RNA or protein), “alteration”, “change”, etc., refers to any detectable “increase / induction” or “decrease / reduction” in the experimental group (e.g., spinach plants with the modifications described herein) compared to the control group (e.g., wild-type spinach plants without modifications).

[0082] As used herein, “reduction”, “lowering”, etc., refer to any detectable reduction in the experimental group (e.g., spinach plants with the modifications described herein) compared to the control group (e.g., wild-type spinach plants without modifications). Therefore, a reduction in protein expression includes any detectable reduction in the total protein level in the sample and can be determined using conventional methods in the art, such as Western blotting and ELISA.

[0083] As used herein, in the context of expression (e.g., RNA or protein), "induced," "increased," etc., refer to any detectable increase in the experimental group (e.g., spinach plants with the modifications described herein) compared to the control group (e.g., wild-type spinach plants without modifications). Therefore, an increase in protein expression includes any detectable increase in the total protein level in a sample and can be determined using conventional methods in the art, such as Western blotting and ELISA.

[0084] The term "single nucleotide polymorphism" or "SNP" refers to a variation in the DNA sequence that occurs when a single nucleotide (A, T, C, or G) in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments, AAGCCTA and AAGCTTA, from different individuals contain a single nucleotide difference. In this case, it can be said that there are two alleles, C and T. Almost all common SNPs have only two alleles.

[0085] In this article, "spinach" or "cultivated spinach" refers to the plant (or seeds from the plant that can grow into a plant) of the species spinach (Spinacia oleracea), as well as parts of that plant, bred for human consumption and possessing favorable agronomic characteristics. This includes any cultivated spinach, such as breeding lines (e.g., backcross lines, inbred lines), cultivars, and varieties (open-pollinated or hybrid varieties). This includes any type of spinach, such as curly-leaf, flat-leaf, or smooth-leaf spinach, or semi-curly-leaf types.

[0086] "Targeted DNA modification" can be used synonymously with targeted DNA mutation and refers to a site-specific modification that introduces an alteration or change in the nucleotide sequence at a specific locus in a plant (e.g., spinach). The targeted DNA modification described herein can be any modification known in the art, such as insertions, deletions, single nucleotide polymorphisms (SNPs), and polynucleotide modifications. Furthermore, the targeted DNA modification at the locus can be located anywhere within the locus, such as the coding region (e.g., exons), non-coding region (introns), regulatory element, or untranslated region of the encoded polypeptide. There are no particular limitations on the type and location of the targeted DNA modification, as long as it results in a change in the expression level or activity of the protein encoded by the S gene. In some embodiments, the targeted DNA modification is the deletion of one or more nucleotides at the locus, preferably a sequential deletion of one or more nucleotides.

[0087] The “unfavorable allele” of a marker is a marker allele that segregates along with an unfavorable plant phenotype, thus providing the benefit of identifying plants that can be removed from breeding programs or planting.

[0088] This article provides statistically significant marker loci that co-segregate with disease resistance traits that confer broad resistance to specific diseases or one or more diseases. Detection of these loci, or other linked loci and resistance genes, can be used as part of marker-assisted selection in breeding programs to produce plants resistant to one or more diseases.

[0089] Drawing genetic maps

[0090] It has long been known that specific genetic loci associated with particular phenotypes, such as disease resistance, can be located in the genome of an organism. By detecting the statistically significant probability of co-segregation with the desired phenotype—that is, marker alleles exhibiting linkage disequilibrium—plant breeders can advantageously use molecular markers to identify desired individuals. By identifying molecular markers or clusters of molecular markers co-segregating with the trait of interest, breeders can rapidly select the desired phenotype by choosing appropriate molecular marker alleles (a process called marker-assisted selection or MAS). Various methods can be used to detect molecular markers or clusters of molecular markers co-segregating with a trait of interest (e.g., disease resistance). The basic idea behind these methods is to detect markers whose alternative genotypes (or alleles) have significantly different average phenotypes. Therefore, the magnitude or significance level of the difference between alternative genotypes (or alleles) is compared between marker loci. The trait gene is inferred to be located at the marker(s) closest to the marker(s) with the largest associated genotypic difference. Two methods for detecting loci of interest are: 1) population-based association analysis (i.e., association localization) and 2) conventional linkage analysis.

[0091] Related Location

[0092] Understanding the extent and patterns of linkage disequilibrium (LD) in the genome is a prerequisite for developing efficient association methods for identifying and locating quantitative trait loci (QTLs). Linkage disequilibrium (LD) refers to the non-random association of alleles within a set of individuals. When observing LD between alleles at linkage loci, it can be measured as LD decay along a specific region of a chromosome. The extent of LD reflects the recombination history of that region. The average LD decay rate in the genome can help predict the number and density of markers needed for genome-wide association studies and provide an estimate of predictable resolution.

[0093] Association or LD mapping aims to identify significant genotype-phenotype associations. It has been developed into a powerful tool for fine mapping in outcrossing species, for example, in humans (Corder et al., (1994) "Protective effect of apolipoprotein-E type-2 allele for late-onset Alzheimer-disease" Nat Genet 7:180-184; Hastbacka et al., (1992) "Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland" Nat Genet 2:204-211; Kerem et al., (1989) "Identification of the cystic fibrosis gene: genetic analysis" Science 245:1073-1080) and maize (Remington et al., (2001) "Structure of linkage disequilibrium and phenotype associations in the maize genome" Proc Natl Acad Sci USA). 98:11479-11484; Thornsberry et al., (2001) "Dwarf8 polymorphisms associate with variation in flowering time" Nat Genet 28:286-289; Flint-Garcia et al., (2003) review "Structure of linkage disequilibrium in plants" Annu Rev Plant Biol. 54:357-374), in which frequent recombination between heterozygotes leads to rapid LD decay. In inbred species, recombination between homozygous genotypes is genetically undetectable, the range of LD is larger (i.e., larger linkage marker segments are co-inherited) and this significantly enhances the ability to detect association localization (Wall and Pritchard (2003) "Haplotype blocks and linkage disequilibrium in the human genome" Nat Rev Genet 4:587-597).

[0094] The recombination and mutation history of a population is associated with mating habits as well as the effective size and age of the population. Larger population sizes increase the likelihood of detecting recombination, while older populations are generally associated with higher levels of polymorphism, both of which lead to a significantly faster rate of LD decay. On the other hand, smaller effective population sizes, such as those that have recently experienced genetic bottlenecks, tend to exhibit a slower rate of LD decay, resulting in broader haplotype conservation (Flint-Garcia et al., (2003) "Structure of linkage disequilibrium in plants" Annu Rev Plant Biol. 54: 357-374).

[0095] Association analysis incorporates quantitative phenotypic scores (e.g., disease tolerance in individual strains, scored from 1 to 9) into the analysis (as opposed to observing only tolerance and the frequency distribution of resistance alleles in intergroup allele distribution patterns). The availability of detailed phenotypic data collected over many years through breeding programs and the environment of a large number of backbone strains provides valuable datasets for genetic marker association mapping analysis. This paves the way for seamless integration between research and application and leverages historically accumulated datasets. However, understanding the relationship between polymorphism and recombination is useful in developing appropriate strategies for efficiently extracting the most information from these resources.

[0096] This association analysis neither generates nor requires any map data, and is independent of map locations. The analysis compares plant phenotypic scores to genotypes at multiple loci. Subsequently, any appropriate map (e.g., a composite map) can be optionally used to aid in observing the distribution of identified QTL markers and / or QTL marker clusters using the previously determined map locations of the markers.

[0097] Conventional Chain Analysis

[0098] Traditional linkage analysis is based on the same principle; however, LD is generated by producing a population from a small number of founders. Founders are selected to maximize the level of polymorphism within the established population, and the level of co-segregation of polymorphic loci with a given phenotype is assessed. Many statistical methods have been used to identify significant marker-trait associations. One such method is interval mapping (Lander and Botstein, Genetics 121:185-199 (1989)), in which the likelihood of a gene controlling the trait of interest being located at each of multiple locations (e.g., spaced 1 cM apart) in the genetic map is tested. A LOD score (logarithm of the likelihood ratio) is calculated for each test location using genotype / phenotype data. When the LOD score exceeds a threshold, there is significant evidence that the location of a gene controlling the trait of interest is located at that location in the genetic map (which would fall between two specific marker loci).

[0099] This article presents statistically significant co-segregating marker loci with disease resistance traits, identified through routine linkage analysis and genome-wide association studies (GWAS). Detection of these loci, or additional linkage loci, can be used in marker-assisted breeding programs to produce disease-resistant plants.

[0100] Activities in marker-assisted breeding programs may include, but are not limited to: selecting from newly bred populations to identify which population has the highest frequency of favorable nucleic acid sequences based on historical genotype and agronomic trait associations; selecting favorable nucleic acid sequences in the offspring of the breeding population; selecting from parental lines based on predictions of offspring performance; and improving lines in germplasm improvement activities based on the presence of favorable nucleic acid sequences.

[0101] markers

[0102] This invention provides molecular markers (i.e., including marker sites and nucleic acids corresponding to (or derived from) these marker sites, such as probes and amplification products) associated with the susceptibility (S) gene in spinach for genotyping of plants, such as Spov3_chr4_93275081, Spov3_chr4_105049870, Spov3_chr4_107241402, and Spov3_chr4_ The molecular markers are 109546568, Spov3_chr4_109698808, Spov3_chr4_112008390, Spov3_chr4_112574123, Spov3_chr4_117783935, Spov3_chr4_118191085, Spov3_chr4_118788268, and Spov3_chr4_121142541. These molecular markers are useful for selecting spinach plants resistant to downy mildew. Therefore, these markers are useful for marker-assisted selection (MAS) and breeding of downy mildew-resistant lines, as well as for the identification of resistant lines. The markers of the present invention can also be used to identify and determine the chromosomal regions corresponding to the S gene. The S gene can be isolated by localized cloning of, for example, a genetic region defined by a pair of markers described herein, or a subsequence of a region defined by and including these markers. Furthermore, the S gene isolated from one organism (such as spinach) can be used in turn to isolate homologous genes of the S gene from other organisms, including a variety of important commercial crops.

[0103] As is known to those skilled in the art, a variety of molecular markers exist. For example, molecular markers may include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), single nucleotide polymorphism (SNP), or simple sequence repeat (SSR).

[0104] Simple sequence repeats (SSRs), or microsatellites, are DNA regions that are repeated in tandem with one or a few bases several to hundreds of times. For example, a dinucleotide repeat might resemble CACACACA, and a trinucleotide repeat might resemble ATGATGATGATG. Simple sequence repeats are thought to arise from slip-mediated errors during DNA replication, repair, and recombination. The length of these repeat sequences varies over time between different cultivars. An example of allelic variation in an SSR might be that allele A is GAGAGAGA (four repeats of the GA sequence) while allele B is GAGAGAGAGA (six repeats of the GA sequence). When an SSR appears in a coding region, its persistence depends on its effect on the structure and function of the encoded protein. Because repeat segments are more susceptible to DNA slip-mediated amplification / deletion, their presence in coding regions is limited by the non-perturbation of the reading frame and tolerance to amino acid extensions amplified in the encoded protein. Of all possible SSRs, trinucleotide repeats or their multiples are more common in coding regions.

[0105] A single nucleotide polymorphism (SNP) is a change in DNA sequence that occurs when a single nucleotide (A, T, C, or G) differs between members of a species (or between pairs of chromosomes in an individual). For example, two sequenced DNA fragments, AAGCCTA and AAGCTTA, from different individuals contain a single nucleotide difference. In this case, it can be said that there are two alleles, C and T.

[0106] Table 1

[0107] Spov3_chr4_93275081 4 93，275，081 C T Spov3_chr4_105049870 4 105，049，870 G A Spov3_chr4_107241402 4 107，241，402 G A Spov3_chr4_109546568 4 109，546，568 G A Spov3_chr4_109698808 4 109，698，808 G A Spov3_chr4_112008390 4 112，008，390 G A Spov3_chr4112574123 4 112，574，123 G A Spov3_chr4_117783935 4 117，783，935 G A Spov3_chr4_118191085 4 118，191，085 G A Spov3_chr4_118788268 4 118，788，268 G A Spov3_chr4_121142541 4 121，142，541 G A

[0108] Spov3_chr4_93275081-SEQ ID NO: 241 (with "C" or "T" at bit 101)

[0109]

[0110] Spov3_chr4_105049870-SEQ ID NO: 242 (with "G" or "A" at position 101)

[0111]

[0112] Spov3_chr4_107241402-SEQ ID NO: 243 (with "G" or "A" at bit 101)

[0113]

[0114] Spov3_chr4_109546568-SEQ ID NO: 244 (with "G" or "A" at bit 101)

[0115]

[0116] Spov3_chr4_109698808-SEQ ID NO: 245 (with "G" or "A" at position 101)

[0117]

[0118] Spov3_chr4_112008390-SEQ ID NO: 246 (with "G" or "A" at bit 101)

[0119]

[0120] Spov3_chr4_112574123-SEQ ID NO: 247 (with "C" or "T" at position 101)

[0121]

[0122]

[0123] Spov3_chr4_117783935-SEQ ID NO: 248 (with "C" or "T" at position 101)

[0124]

[0125] Spov3_chr4_118191085-SEQ ID NO: 249 (with "G" or "A" at position 101)

[0126]

[0127] Spov3_chr4_118788268-SEQ ID NO: 250 (with "G" or "A" at position 101)

[0128]

[0129] Spov3_chr4_121142541-SEQ ID NO: 251 (with "G" or "A" at bit 101)

[0130]

[0131] Linkage markers

[0132] Those skilled in the art will recognize that additional molecular markers can be identified within the intervals defined by the aforementioned marker pairs (e.g., Spov3_chr4_93275081 and Spov3_chr4_121142541). These markers are also genetically linked to the S gene and are within the scope of this invention. The markers can be identified by any of a variety of genetic or physical mapping techniques. Methods for determining whether a marker is genetically linked to the S gene are known to those skilled in the art and include, for example, interval mapping (Lander and Botstein, (1989) Genetics 121:185), regression mapping (Haley and Knott, (1992) Heredity 69:315), or MQM mapping (Jansen, (1994) Genetics 138:871). Furthermore, in the context of this invention, physical mapping techniques such as chromosome walking, contiguous group mapping, and assembly can be used to identify and isolate additional sequences that can be used as markers.

[0133] Chromosomal regions

[0134] Chromosomal regions associated with disease resistance traits are provided. Various methods can be used to identify chromosomal regions. The boundaries of these chromosomal regions are defined to include markers linked to one or more genes controlling the trait of interest. In other words, chromosomal regions are defined such that any marker located within that region (including terminal markers defining the region boundaries) can be used as a marker for the disease resistance trait.

[0135] Conversely, for example, if two closely adjacent markers exhibit co-segregation with the desired phenotypic trait, it is sometimes unclear whether these markers recognize the same gene or two different genes or multiple genes. In any case, it is not necessary to know how many genes reside within a specific physical / genomic region for the manufacture or implementation of the contents proposed in this invention.

[0136] The region of chromosome 4 may include any of the markers identified in this paper as being associated with the DM resistance trait. These markers include “T” at Spov3_chr4_93275081 (reference sequence SEQ ID NO: 241, position 101), “A” at Spov3_chr4_105049870 (reference sequence SEQ ID NO: 242, position 101), “A” at Spov3_chr4_107241402 (reference sequence SEQ ID NO: 243, position 101), “A” at Spov3_chr4_109546568 (reference sequence SEQ ID NO: 244, position 101), “A” at Spov3_chr4_109698808 (reference sequence SEQ ID NO: 245, position 101), and “T” at Spov3_chr4_112008390 (reference sequence SEQ ID NO: 245, position 101). The following are examples of sequences with different positions: "A" at position 101 of SEQ ID NO: 246, "T" at position 101 of SEQ ID NO: 247 (reference sequence), "T" at position 101 of SEQ ID NO: 248 (reference sequence), "A" at position 101 of SEQ ID NO: 249 (reference sequence), "A" at position 101 of SEQ ID NO: 250 (reference sequence), or "A" at position 101 of SEQ ID NO: 251 (reference sequence). Any marker located within these intervals can be used as a marker of DM resistance and can be used in the context of the methods proposed herein to identify and / or select plants resistant to DM, whether newly conferred or enhanced compared to control plants.

[0137] Chromosomal regions can also be defined by markers linked to disease resistance genes (showing linkage disequilibrium with those genes), and in the context of association studies, R 2 It is a commonly used measure of linkage disequilibrium (LD). If the LD between a chromosome 4 marker locus in the interval of interest and another closely adjacent chromosome 4 marker locus is r... 2 If the value is greater than 1 / 3 (Ardlie et al., Nature Reviews Genetics 3: 299-309 (2002)), then these sites are in linkage disequilibrium.

[0138] Detection of marker sites

[0139] Markers corresponding to genetic polymorphisms among members of a population can be detected by a variety of methods recognized in the art (e.g., restriction fragment length polymorphism, isoenzyme markers, allele-specific hybridization (ASH), amplified variable sequences in the plant genome, autonomous sequence duplication, simple sequence repeats (SSR), single nucleotide polymorphisms (SNPs) or amplified fragment length polymorphisms (AFLPs)).

[0140] The detection of most genetic markers relies on more than one property of the nucleic acid. For example, some techniques for detecting genetic markers utilize the hybridization of probe nucleic acids with nucleic acids corresponding to the genetic marker. Hybridization forms include, but are not limited to, solution-phase, solid-phase, mixed-phase, or in situ hybridization assays. Restriction fragment length polymorphism (RFLP) markers are detected by hybridization with restriction-digested genomic DNA using a probe (typically a sub-fraction of the nucleic acid to be tested or a synthetic oligonucleotide corresponding to the sub-fraction). Restriction enzymes are selected to provide at least two alternative (or polymorphic) lengths of restriction fragments in different individuals and are typically strain-specific. Identifying (one or more) restriction enzymes that produce the fragments providing information for each hybridization is a simple and well-known step in the art. After separation by length in a suitable matrix (e.g., agarose) and transfer to a membrane (e.g., nitrocellulose, nylon), the marker probe is hybridized under conditions that result in balanced binding of the probe to the target, followed by removal of excess probe by washing.

[0141] Nucleic acid probes targeting marker sites can be cloned and / or synthesized. Suitable detectable tags for use with nucleic acid probes include any combination detectable by spectroscopic, radioisotope, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful tags include biotin for streptavidin conjugate staining of the marker, magnetic beads, fluorescent dyes, radioactive tags, enzymes, and colorimetric tags. Other tags include ligands, chemiluminescent agents, and enzymes that bind to antibodies labeled with fluorescent chromophores. Marker tags can be readily implemented, for example, using PCR primers that apply the marker to the marker site.

[0142] The hybridization probe is then typically detected using autoradiography or other similar detection techniques (e.g., autofluorescence, liquid scintillation counter, etc.). Examples of specific hybridization schemes are widely available in this field; see, for example, Berger, Sambrook, Ausubel, all cited above.

[0143] Amplified variable sequences refer to amplified sequences of plant genomes that exhibit high variability in nucleic acid residues among members of the same species. All organisms possess variable genome sequences, and each organism (except clones) has a distinct set of variable sequences. The presence of specific variable sequences, once identified, can be used to predict phenotypic traits. Preferably, DNA from the plant is used as the template for amplification, and primers are positioned flanking the variable sequence of the DNA. The variable sequence is amplified and sequenced.

[0144] In vitro amplification techniques are well known in this field. Examples of techniques sufficient to guide technicians through these in vitro methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicaase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA), as seen in Berger, Sambrook, and Ausubel (cited above), and Mullis et al., (1987) U.S. Patent No. 4,683,202; PCR Protocols, A Guide to Methods and Applications (edited by Innis et al.), Academic Press Inc., San Diego Academic Press Inc., San Diego, Calif. (1990) (Innis); Arnheim and Levinson, (Oct. 1, 1990) C&E7V 36-47; The Journal of NIH Research (1991) 3: 81-94; (Kwoh et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874; Lomeli et al., (1989) J. Clin. Chem 35:1826; Landegren et al., (1988) Science 241:1077-1080; Van Brunt, (1990) Biotechnology 8:291-294; Wu and Wallace, (1989) Gene 4:560; Barringer et al., (1990) Gene 89:117 and Sooknanan and Malek, (1995) Biotechnology 13:563-564. A modified method for cloning nucleic acids by in vitro amplification is described in Wallace et al., US Patent No. 5,426,039. A modified method for amplifying large nucleic acids by PCR is described in Cheng et al., (1994) Nature. Summary in 369:684 and its references, which describes the production of PCR amplicons up to 40kb. Technicians will understand that virtually any RNA can be converted into double-stranded DNA suitable for restriction digestion, PCR amplification, and sequencing using reverse transcriptase and polymerase. See Ausubel, Sambrook, and Berger, all cited above.

[0145] For example, oligonucleotides used as primers and nucleic acid sequence probes in amplification reactions are typically chemically synthesized according to the solid-phase phosphoramidite method as described in Beaucage and Caruthers, (1981) Tetrahedron Lett. 22: 1859, or can be simply ordered commercially.

[0146] Alternatively, autonomous sequence replication can be used to identify genetic markers. Autonomous sequence replication refers to a nucleic acid amplification method that uses the exponential replication of the target nucleic acid sequence in vitro under essentially isothermal conditions by employing the activities of three enzymes involved in retroviral replication: (1) reverse transcriptase, (2) RNase H, and (3) DNA-dependent RNA polymerase (Guatelli et al., (1990) Proc Natl Acad Sci USA 87:1874). By mimicking the RNA replication strategy of retroviruses through cDNA intermediates, this reaction accumulates copies of the original target cDNA and RNA.

[0147] As described above, there are various types of molecular tags, including amplified fragment length polymorphisms (AFLP), allele-specific hybridization (ASH), single nucleotide polymorphisms (SNPs), simple sequence repeats (SSRs), and isoenzyme markers. The methods of using different types of molecular markers are known to those skilled in the art. The markers of this invention include simple sequence repeats and single nucleotide polymorphisms.

[0148] Reagent test kit

[0149] A kit is also provided for screening germplasm containing the markers of the present invention. The kit includes the polynucleotides of the present invention, fragments thereof, or complementary sequences, used as probes or primers to detect markers of the S gene. Instructions for use of the polynucleotides, as well as buffers and / or other solutions, may also be provided to facilitate the use of the polynucleotides.

[0150] Marker-assisted plant selection and breeding

[0151] The spinach plants of this invention can be used in plant breeding programs. The goal of plant breeding is to combine various desired agronomic traits in a single variety or hybrid. For field crops, these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to cold or frost, shortened crop maturity time, higher yield, and better agronomic quality. In the case of mechanically harvested multiple crops, uniform plant characteristics are advantageous, such as germination and emergence, growth rate, maturity, and plant and ear height. Conventional plant breeding is an important tool for developing new, improved commercial crops. This invention includes a method for producing plants by crossing a first parent plant with a second parent plant, wherein one or both parent plants exhibit the phenotypes described herein.

[0152] Plant breeding techniques known in this field and used in plant breeding programs include, but are not limited to, recurrent selection, group selection, mixed selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism (RFLP) enhancement selection, genetic marker enhancement selection, haploid doubling, and transformation. Common combinations of these techniques are also discussed. The marker loci and alleles provided herein can be used in marker-assisted selection (MAS) breeding. The closer the linkage of a marker to a DNA locus affecting a phenotype (e.g., disease resistance), the more reliable the marker is in MAS, as the likelihood of recombination events unlinked to the locus decreases. Markers containing causal polymorphisms related to the trait or located within the coding sequence of the gene causing the trait are advantageous because recombination is not expected between them and the DNA sequence causing the phenotype. However, markers do not necessarily contain or correspond to incidental polymorphisms to be effective in MAS. In fact, most MAS breeding uses only markers linked to causal mutations.

[0153] Developing molecular markers in crop species can improve plant breeding efficiency through MAS (Magnetic Analogous Sequence). Genetic markers are used to identify plants that contain a desired genotype at more than one locus and are expected to transmit the desired genotype along with the desired phenotype to their progeny plants. Genetic markers can also be used to identify plants that contain a desired genotype at one locus or at multiple linked or unlinked loci (e.g., haplotypes) and are expected to transmit the desired genotype and desired phenotype to their progeny plants.

[0154] Generally, MAS uses polymorphic markers that have been identified as having a significant probability of cosegregation with the desired trait. These markers are presumed to be located near one or more genes that provide the plant with its desired phenotype and are considered indicators of the desired trait.

[0155] Identification of plants or germplasm containing one or more marker loci linked to one or more desired traits provides the basis for MAS (Magnetic Attraction System). Plants containing favorable markers or favorable alleles are selected, while plants containing markers or alleles negatively correlated with the desired trait are eliminated. The desired markers and / or alleles can be introgressed into plants with the desired genetic background (e.g., backbone or exogenous), thereby producing introgressed plants or germplasm with the desired trait. In some aspects, it may be considered to select and / or introgress multiple markers of the desired trait sequentially or simultaneously. There is no limitation on the combination of markers selected in a single plant, and it may include any combination of markers disclosed herein or any markers linked to the markers disclosed herein.

[0156] This invention provides a method for identifying disease-resistant spinach plants with altered S gene expression by identifying plants with specific alleles, wherein the specific alleles are, for example, one or more of the markers Spov3_chr4_93275081, Spov3_chr4_105049870, Spov3_chr4_107241402, Spov3_chr4_109546568, Spov3_chr4_109698808, Spov3_chr4_112008390, Spov3_chr4_112574123, Spov3_chr4_117783935, Spov3_chr4_118191085, Spov3_chr4_118788268, and Spov3_chr4_121142541. Similarly, by identifying plants lacking the required alleles, plants that do not possess disease resistance can be identified and, for example, removed from subsequent hybridization.

[0157] MAS is a powerful shortcut for selecting desired phenotypes and infiltrating desired traits into cultivars (e.g., infiltrating desired traits into backbone lines). MAS is readily used in high-throughput molecular analysis methods that can rapidly extract markers of interest from large quantities of plant or germplasm genetic material at a much higher cost-effectiveness than culturing and observing visible traits in plants.

[0158] When populations are segregated based on multiple sites that influence one of multiple traits (e.g., multiple sites involved in disease resistance), MAS is even more efficient than phenotypic screening because all sites can be evaluated together in a single DNA sample.

[0159] Another use of MAS in plant breeding is to assist in the restoration of the genotype of a recurrent parent through backcrossing. Backcrossing is the process of crossing offspring with one of their parents. Backcrossing is typically performed to introduce one or more loci from the donor parent into other desired genetic backgrounds from the recurrent parent. The more backcross cycles performed, the greater the genetic contribution of the recurrent parent to the resulting variety. This is often necessary because the donor parent plant may be disadvantaged in other ways, such as low yield or low fertility. Conversely, varieties obtained through enhanced breeding programs may possess superior yield, fertility, etc., lacking only one desired trait. As understood by those skilled in the art, backcrossing can be performed to select for or eliminate traits.

[0160] Introgression of one or more desired loci from a donor line into another line is achieved through repeated backcrosses with a recurrent parent and selection to retain one or more loci from the donor parent. Sex-determining markers are examined in the offspring, and those offspring possessing one or more desired markers are selected for the next step. Alternatively, one or more markers may be examined in the offspring to select plants with the parent's genotype. The invention anticipates that trait introgression may require more than one generation, wherein offspring are crossed with or self-crossed with a recurrent parent. Selection is based on the presence of one or more sex-determining markers and may also be based on the recurrent parent's genotype, wherein selection is based on genetic markers and / or phenotypes. In another embodiment, markers may be used in conjunction with other markers, advantageously at least one on each chromosome of the spinach genome, to track introgression in the skeletal germplasm.

[0161] Targeted cloning

[0162] As described above, the molecular markers of the present invention can be used, such as Spov3_chr4_93275081, Spov3_chr4_105049870, Spov3_chr4_107241402, Spov3_chr4_109546568, Spov3_chr4_109698808, Spov3_chr4_112008390, and Spov3_chr4_11257. 4123, Spov3_chr4_117783935, Spov3_chr4_118191085, Spov3_chr4_118788268, and Spov3_chr4_121142541, and their homologous nucleic acids, can be used to identify additional linkage marker sites, which can be cloned using recognized procedures, such as those described in detail above by Ausubel, Berger, and Sambrook. Similarly, these markers and genes, as well as any additional identified linkage molecular markers, can be used to physically isolate (e.g., by cloning) nucleic acids associated with markers that promote fertility restoration. These nucleic acids, i.e., those linked to the markers, have multiple uses, including as genetic markers for identifying additional markers in subsequent applications of marker-assisted selection (MAS). These nucleic acids may also include the S gene itself.

[0163] These nucleic acids are first identified by their genetic linkage with the markers of the present invention. The isolation of the nucleic acids of interest is achieved by any number of methods as discussed in detail in references such as Ausubel, Berger and Sambrook (ibid.), and Clark, Ed. (1997) Plant Molecular Biology: A Laboratory Manual, Springer-Verlag, Berlin, etc.

[0164] For example, “localized gene cloning” uses the proximity of genetic markers to physically define isolated chromosomal segments linked to a gene. Isolated chromosomal segments can be generated by well-known methods, such as digesting chromosomal DNA with one or more restriction enzymes, amplifying chromosomal regions in polymerase chain reaction (PCR) or alternative amplification reactions, or by direct Sanger sequencing. The digested or amplified segments are typically ligated into vectors suitable for insertion replication and optional expression, such as plasmids, granules, bacteriophages, artificial chromosomes, etc. Markers adjacent to the open reading frame (ORF) associated with the phenotypic trait can hybridize with the DNA clone to identify the clone localized by the ORF. If the markers are further apart, segments containing the ORF are identified by successive rounds of screening and isolation of clones containing consecutive DNA sequences (“contigs”). Protocols sufficient to guide technicians in isolating clones associated with linkage markers can be found in Berger, Sambrook, and Ausubel, all cited above.

[0165] Separated chromosomal regions and separated S genes

[0166] This invention provides a chromosomal region containing S genes involved in disease resistance. The gene is located in the region defined by two markers of this invention (Spov3_chr4_93275081 (SEQ ID NO: 241) and Spov3_chr4_121142541 (SEQ ID NO: 251), each marker being genetically linked to the gene. The chromosomal region may contain more than one disease resistance-associated ORF and may be cloned on more than one independent vector, for example, depending on the size of the chromosomal region. For example, in this invention, three genes were identified within the region sandwiched between the SNP markers Spov3_chr4_93275081 and Spov3_chr4_121142541. In summary, three groups of S genes were identified that function at different stages of infection: the early pathogen formation stage, the stage regulating host defense, and the pathogen survival stage (Annu RevPhytopathol. 2014; 52: 551-81).

[0167] This invention specifically provides isolated nucleic acids, such as DNA, RNA, homologs, paralogs, and orthologs, and / or chimeras thereof, including S gene polynucleotides and their encoded proteins. This includes naturally occurring sequences as well as synthetic variants and homologs.

[0168] This invention includes clathrin aptamer subunit polypeptides. The terms "clathrin aptamer subunit polypeptide" and "clathrin aptamer subunit protein," used interchangeably herein, refer to one or more polypeptides associated with disease resistance and are sufficiently identical to the spinach clathrin aptamer subunit polypeptide of SEQ ID NO: 6. Various clathrin aptamer subunit polypeptides may be considered.

[0169] This invention includes polypeptides containing triangular pentapeptide repeats. The terms "polypeptide containing triangular pentapeptide repeats" and "protein containing triangular pentapeptide repeats" as used interchangeably herein refer to one or more polypeptides associated with disease resistance and are sufficiently identical to the spinach polypeptide containing triangular pentapeptide repeats of SEQ ID NO: 98. Various polypeptides containing triangular pentapeptide repeats are contemplated.

[0170] This invention includes a resistance polypeptide against *Pseudomonas syringae* 2-like (RPS2-like) peptides. The terms "RPS2-like peptide" and "RPS2-like protein" may be used interchangeably herein to refer to one or more peptides associated with disease resistance and to have sufficient identity with the spinach RPS2-like peptide of SEQ ID NO: 174. Various RPS2-like peptides may be considered.

[0171] As used herein, “sufficient identity” means a sequence identity with the parent amino acid sequence of at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, sequence identity refers to the full-length sequence of the polypeptide. The term “about” in the context of sequence identity percentages herein means + / - 1.0%.

[0172] "Fragment" or "bioactive portion" includes a polypeptide or polynucleotide fragment containing a sequence that has sufficient identity with a subunit of the clathrin aptamer, a triangular pentapeptide repeat, or an RPS2-like resistant polypeptide or polynucleotide, respectively.

[0173] As used herein, “variant” refers to a protein or polypeptide whose amino acid sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the parent amino acid sequence.

[0174] In some embodiments, the clathrin aptamer subunit polypeptide, the polypeptide containing a triangular pentapeptide repeat, or the RPS2-like resistance polypeptide comprises the same as SEQ ID NO: 6, SEQ ID NO: 98, or SEQ ID NO: 98. The full length or fragment of the amino acid sequence of NO: 174 has an amino acid sequence with at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of identity.

[0175] Sequences homologous to the sequences provided herein (i.e., having significant sequence identity or similarity) are also an aspect of this invention. Homologous sequences can originate from any plant, including monocots and dicots, particularly agriculturally important plant species, including but not limited to crops such as soybeans, wheat, corn, potatoes, cotton, rapeseed (including canola), sunflowers, alfalfa, clover, sugarcane, and lawn crops; or fruits and vegetables such as bananas, blackberries, blueberries, strawberries and raspberries, cantaloupes, carrots, cauliflower, coffee, cucumbers, eggplants, grapes, honeydew melons, lettuce, mangoes, cantaloupes, onions, papayas, peas, peppers, pineapples, squash, spinach, pumpkins, sweet corn, tobacco, tomatoes, tomatoes, watermelons, Rosaceae fruits (e.g., apples, peaches, pears, cherries, and plums) and Brassica vegetables (e.g., broccoli, cabbage, cauliflower, Brussels sprouts, and turnips). Other crops, including fruits and vegetables whose phenotypes can be altered and which contain homologous sequences, include basil, barley; rye; millet; sorghum; currants; avocados; hops; citrus fruits such as oranges, lemons, grapefruits, and tangerines, artichokes, and cherries; chicory; leeks; root vegetables such as calathea, beets, cassava, turnips, radishes, yams, and sweet potatoes; and legumes. Furthermore, homologous sequences can originate from plants that are evolutionarily related to crop plants but may not yet be used as crops.

[0176] Homologous sequences can include orthologous or paralogous sequences. Those skilled in the art are familiar with several different methods for identifying and defining these functionally homologous sequences. Three conventional methods for defining orthologs and paralogs are described; orthologs, paralogs, or homologs can be identified by one or more of these methods.

[0177] Orthologs and paralogs are evolutionarily related genes with similar sequences and functions. Orthologs are structurally related genes originating from speciation events in different species. Paralogs are structurally related genes originating from amplification events within a single species.

[0178] Within a single plant species, gene duplication can produce two copies of a specific gene, resulting in two or more genes with similar sequences and generally similar functions, also known as paralogs. Therefore, paralogs are similar genes formed through amplification within the same species. When analyzing the phylogeny of gene families, paralogs are often clustered together or in the same branch (a group of similar genes), using analytical procedures such as CLUSTAL (Thompson et al., (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al., (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified using pairwise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25: 351-360).

[0179] For example, branches of very similar MADS domain transcription factors in Arabidopsis thaliana have the same function in terms of flowering time (Ratcliffe et al., (2001) Plant Physiol. 126: 122-13), and a group of very similar AP2 domain transcription factors in Arabidopsis thaliana are involved in plant tolerance to frost damage (Gilmour et al., (1998) Plant J. 16: 433-442). Analyzing groups of similar genes with similar functions that fall into the same branch can yield branch-specific subsequences. These subsequences, also called common sequences, can be used not only to identify sequences within each branch but also to determine the function of these genes; genes within a branch can contain paralogous or orthologous sequences with the same function (see also, for example, Mount (2001), in Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, page 543.).

[0180] Speciation, the process by which a new species emerges from a parent species, can also generate two or more genes with similar sequences and functions. These genes are called orthologs, which typically have the same function in their host plants and are usually interchangeable between species without loss of function. Because plants share a common ancestor, many genes in any plant species have corresponding orthologs in another plant species. After constructing a phylogenetic tree of a species' gene family using software such as CLUSTAL (Thompson et al., (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al., (1996), as described above), potential orthologous sequences can be placed into the phylogenetic tree, and their relevance to genes from the species of interest can be determined. Orthologous sequences can also be identified using a reverse BLAST strategy. After identifying orthologous sequences, the function of the ortholog can be deduced from the function already determined for the reference sequence.

[0181] Orthologous genes from different organisms possess highly conserved functions and are often substantially identical (Lee et al., (2002) Genome Res. 12: 493-502; Remm et al., (2001) J. Mol. Biol. 314: 1041-1052). Paralogous genes derived through gene replication and differentiation may retain similar functions of the encoded proteins. In such cases, paralogs can be used interchangeably for certain embodiments of the present invention (e.g., transgenic expression of coding sequences).

[0182] Exemplary sequences homologous to the subunit polynucleotides (SEQ ID NOs: 4 or 5) of the spinach clathrin aptamer are shown in SEQ ID NOs: 7-63, 252-293 and summarized in Tables 2, 5, 6 and 7. SEQ ID NO: 6 relates to the subunit protein sequence of the spinach clathrin aptamer, and homologous protein sequences from other plant species are shown in SEQ ID NOs: 64-92. Other homologous protein sequences are given in SEQ ID NOs: 294-315.

[0183] Exemplary sequences homologous to the polynucleotide containing the triangular pentapeptide repeat sequence in spinach (SEQ ID NOs: 96 or 97) are shown in SEQ ID NOs: 99-144, 317-325 and summarized in Tables 3, 5, 6 and 7. SEQ ID NO: 98 relates to a protein sequence containing the triangular pentapeptide repeat sequence in spinach, and homologous protein sequences from other plant species are shown in SEQ ID NOs: 145-168. Other homologous protein sequences are shown in SEQ ID NOs: 326-330.

[0184] Exemplary sequences homologous to spinach RPS2-like resistance polynucleotides (SEQ ID NOs: 172 or 173) are shown in SEQ ID NOs: 175-218, 331-332, and summarized in Tables 4 and 6. Homologous protein sequences are given in SEQ ID NOs: 219-240 and 333.

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] Altering the activity of the protein encoded by the S gene

[0195] In some embodiments, the present invention may include, possibly at certain developmental stages or in tissues, regulating the S gene by transforming plant cells with an expression cassette containing a polynucleotide that alters protein expression, thereby altering, inducing, reducing, or eliminating the activity of the protein encoded by the S gene. The polynucleotide can directly alter protein expression by affecting the transcription or translation of messenger RNA, or indirectly alter protein expression by encoding polypeptides that induce, reduce, or inhibit transcription or translation. Methods for altering, inducing, reducing, inhibiting, or eliminating gene expression in plants are well known in the art, and any such method can be used in the present invention to alter the expression of the protein encoded by the S gene. Various methods can be used to reduce or eliminate, or otherwise alter, the activity of the protein encoded by the S gene. Furthermore, more than one method can be used to alter the activity of the protein encoded by the S gene.

[0196] 1. Polynucleotide-based methods:

[0197] In some embodiments, plants are transformed with a polynucleotide expression cassette capable of expressing the protein encoded by the S gene repressor of the present invention. As used herein, the term "expression" refers to the biosynthesis of a gene product, including transcription and / or translation of said gene product. For example, for the purposes of the present invention, a polynucleotide expression cassette capable of expressing the expression of at least one protein encoded by the S gene repressor is an expression cassette capable of producing an RNA molecule that generates the transcription and / or translation of at least one protein encoded by the S gene repressor of the present invention. "Expression" or "production" of a protein or polypeptide from a DNA molecule means the transcription and translation of the coding sequence to produce the protein or polypeptide, while "expression" or "production" of a protein or polypeptide from a DNA molecule means the translation of an RNA coding sequence to produce the protein or polypeptide.

[0198] The following are examples of polynucleotides that suppress the expression of proteins encoded by the S gene.

[0199] i. Justice Suppression / Co-suppression

[0200] In some embodiments of the invention, suppression of protein expression encoded by the S gene can be achieved through either positive or negative suppression. For negative suppression, the expression cassette is designed to express an RNA molecule corresponding to all or part of the messenger RNA encoding the protein in the "positive" direction. Overexpression of the RNA molecule can lead to a decrease in the expression of the endogenous gene. Therefore, multiple plant lines transformed with the negative suppression expression cassette can be screened to identify lines exhibiting the greatest protein expression suppression.

[0201] The polynucleotide used for co-repression can correspond to all or a portion of the sequence encoding the protein, all or a portion of the 5' and / or 3' untranslated region of the transcript, or all or a portion of the coding sequence and untranslated region of the transcript encoding the protein. In some embodiments where the polynucleotide contains all or a portion of the coding region of the protein, the expression cassette is designed to eliminate the start codon of the polynucleotide so that the protein product is not translated.

[0202] Co-suppression can be used to suppress the expression of plant genes, resulting in plants where protein levels are undetectable for the proteins encoded by those genes. See, for example, Broin et al., (2002) Plant 14: 1417-1432. Co-suppression can also be used to suppress the expression of multiple proteins in the same plant. See, for example, U.S. Patent 5,942,657. The method of using co-suppression to suppress the expression of endogenous genes in plants is described in Flavell et al., (1994) Proc. Natl. Acad. Sci. USA 91: 3490-3496; Jorgensen et al., (1996) Plant Mol. Biol. 31: 957-973; Johansen and Carrington, (2001) Plant Physiol. 126: 930-938; Broin et al., (2002) Plant Cell 14: 1417-1432; Stoutjesdijk et al., (2002) Plant Physiol. 129: 1723-1731; Yu et al., (2003) Phytochemistry 63: 753-763; and U.S. Patents 5,034,323, 5,283,184 and 5,942,657. The efficiency of co-repression can be improved by including a poly-dT region at the 3' of the sense sequence of the expression cassette and at the 5' of the polyadenylation signal. See U.S. Patent Publication 20020048814. Typically, such nucleotide sequences have high sequence identity with the transcript of the endogenous gene, advantageously greater than about 65%, more advantageously greater than about 85%, and most advantageously greater than about 95%. See U.S. Patents 5,283,184 and 5,034,323.

[0203] ii. Antisense inhibition

[0204] In some embodiments of the present invention, suppression of the expression of proteins encoded by the S gene can be achieved through antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule that is wholly or partially complementary to the messenger RNA encoding the protein. Overexpression of the antisense RNA molecule can lead to a decrease in the expression of the endogenous gene. Therefore, multiple plant lines transformed with the antisense suppression expression cassette can be screened to identify the lines exhibiting the greatest protein expression suppression.

[0205] The polynucleotide used for antisense repression can correspond to all or part of the complementary sequence of the sequence encoding the protein, all or part of the complementary sequence of the 5' and / or 3' untranslated region of the transcript, or all or part of the complementary sequence of the coding sequence and untranslated region of the transcript encoding the protein. Furthermore, the antisense polynucleotide can be completely complementary to the target sequence (i.e., 100% identity with the complementary sequence of the target sequence) or partially complementary (i.e., less than 100% identity with the complementary sequence of the target sequence). Antisense repression can be used to suppress the expression of multiple proteins in the same plant. See, for example, U.S. Patent 5,942,657. Additionally, a portion of the antisense nucleotide can be used to disrupt the expression of the target gene. Typically, sequences having at least 50, 100, 200, 300, 400, 450, 500, or 550 nucleotides or more can be used. Methods for using antisense repression to suppress the expression of endogenous genes in plants are described, for example, in Liu et al., (2002) Plant Physiol. 129: 1732-1743 and U.S. Patents 5,759,829 and 5,942,657. The efficiency of antisense repression can be improved by including a poly-dT region at the 3' of the antisense sequence and the 5' of the polyadenylation signal in the expression cassette. See U.S. Patent Publication 20020048814.

[0206] iii. Double-stranded RNA interference

[0207] In some embodiments of the present invention, inhibition of the expression of proteins encoded by the S gene can be achieved through double-stranded RNA (dsRNA) interference. For dsRNA interference, expression of similar sense RNA molecules that co-inhibit the aforementioned sense RNA molecules, and antisense RNA molecules that are fully or partially complementary to the sense RNA molecules, in the same cell leads to inhibition of the expression of the corresponding endogenous messenger RNA.

[0208] Expression of sense and antisense molecules can be achieved by designing expression cassettes to contain both sense and antisense sequences. Alternatively, separate expression cassettes can be used for the sense and antisense sequences. Multiple plant lines transformed with dsRNA interference cassettes or multiple cassettes can then be screened to identify lines exhibiting the greatest protein expression repression. Methods for using dsRNA interference to suppress the expression of endogenous genes in plants are described in Waterhouse et al., (1998) Proc. Natl. Acad. Sci. USA 95: 13959-13964, Liu et al., (2002) Plant Physiol. 129: 1732-1743, and WO 99 / 49029, WO 99 / 53050, WO 99 / 61631, and WO 00 / 49035.

[0209] iv. Hairpin RNA interference and intron-containing hairpin RNA interference

[0210] In some embodiments of the present invention, suppression of the expression of the protein encoded by the S gene can be achieved by hairpin RNA (hpRNA) interference and intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient in suppressing endogenous gene expression. See Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4: 29-38 and the references cited therein.

[0211] For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes to itself to form a hairpin structure containing a single-stranded loop region and a base-pairing stem. The base-pairing stem region contains a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene to be suppressed, and an antisense sequence that is all or part complementary to the sense sequence. Alternatively, the base-pairing stem region may correspond to a portion of the promoter sequence controlling the expression of the gene to be suppressed. Therefore, the base-pairing stem region of the molecule typically determines the specificity of the RNA interference. hpRNA molecules are highly efficient at suppressing endogenous gene expression, and the RNA interference they induce can be inherited by the plant's offspring. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al., (2002) Plant Physiol. 129: 1723-1731; and Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4: 29-38. Methods using hpRNA interference to suppress or silence gene expression are described, for example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al., (2002) Plant Physiol. 129: 1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4: 29-38; Pandolfini et al., BMC Biotechnology 3: 7, and U.S. Patent Publication 2003 / 0175965. A transient test of the efficiency of hpRNA constructs in silencing gene expression in vivo was described by Panstruga et al., (2003) Mol. Biol. Rep. 30: 135-140.

[0212] For ihpRNA, the interfering molecule has the same overall structure as hpRNA, but the RNA molecule additionally contains introns that can be spliced ​​in cells expressing ihpRNA. The use of introns minimizes the loop size in the hairpin RNA molecule after splicing, which improves the interference efficiency. See, for example, Smith et al., (2000) Nature 407:319-320. In fact, Smith et al. showed that ihpRNA-mediated interference 100% suppressed endogenous gene expression. Methods using ihpRNA interference to suppress endogenous plant gene expression are described in, for example, Smith et al., (2000) Nature 407:319-320; Wesley et al., (2001) Plant J.27:581-590; Wang and Waterhouse, (2001) Curr. Opin. Plant Biol.5:146-150; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.4:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295 and U.S. Patent Publication 2003 / 0180945.

[0213] Expression cassettes for hpRNA interference can also be designed so that the sense and antisense sequences do not correspond to endogenous RNA. In this embodiment, the sense and antisense sequences are located on either side of a loop sequence containing nucleotide sequences that correspond to all or part of the endogenous messenger RNA of the target gene. Therefore, the loop region determines the specificity of the RNA interference. See, for example, WO 02 / 00904; Mette et al., (2000) EMBO J 19: 5194-5201; Matzke et al., (2001) Curr. Opin. Genet. Devel. 11: 221-227; Scheid et al., (2002) Proc. Natl. Acad. Sci. USA 99: 13659-13662; Aufsaftz et al., (2002) Proc. Nat'l. Acad. Sci. 99(4): 16499-16506; Sijen et al., Curr. Biol. (2001) 11: 436-440).

[0214] v. Amplicon-mediated interference

[0215] Amplicon expression cassettes contain plant virus-derived sequences that contain all or part of the target gene, but typically not the full gene of the natural virus. The viral sequence present in the transcript of the expression cassette allows the transcript to direct its own replication. The transcript generated by the amplicon can be positive or negative relative to the target sequence (i.e., the messenger RNA). Methods for using amplicons to suppress the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J.16:3675-3684, Angell and Baulcombe, (1999) Plant J.20:357-362, and U.S. Patent No. 6,635,805.

[0216] vi. ribozyme

[0217] In some embodiments, the polynucleotide expressed by the expression cassette of the present invention is catalytic RNA or a ribozyme with protein-messenger RNA specificity. Therefore, the polynucleotide leads to the degradation of endogenous messenger RNA, thereby reducing the expression of the protein encoded by the S gene. This method is described, for example, in U.S. Patent 4,987,071.

[0218] vii. Small interfering RNA or microRNA

[0219] In some embodiments of the present invention, suppression of the expression of the protein encoded by the S gene can be achieved through RNA interference caused by the expression of a gene encoding a microRNA (miRNA). miRNA is a regulator composed of approximately 22 ribonucleotides. miRNAs are highly effective in suppressing the expression of endogenous genes. See, for example, Javier et al., (2003) Nature 425:257-263.

[0220] For miRNA interference, the expression cassette is designed to express an RNA molecule that mimics an endogenous miRNA gene. The miRNA gene encodes an RNA in a hairpin structure containing a 22-nucleotide sequence complementary to another endogenous gene (the target gene). For the repression of S gene expression, the 22-nucleotide sequence is selected from the transcript sequence and contains 22 nucleotides of said sequence in the sense direction and 21 nucleotides of a corresponding antisense sequence complementary to the sense sequence. The miRNA molecules are highly efficient in repressing endogenous gene expression, and the RNA interference they induce can be inherited by the plant's offspring.

[0221] 2. Peptide-based gene expression inhibition

[0222] In one embodiment, a zinc finger protein encoding a polynucleotide binds to the S gene encoding a polypeptide, thereby reducing the expression of the S gene. In a particular embodiment, the zinc finger protein binds to the regulatory region of the S gene. In other embodiments, the zinc finger protein binds to the messenger RNA encoding the polypeptide and prevents its translation. Methods for selecting sites for zinc finger protein targeting have been described, for example, in U.S. Patent 6,453,242, and methods for using zinc finger proteins to suppress gene expression in plants have been described, for example, in U.S. Patent Publication 2003 / 0037355.

[0223] 3. Peptide-based inhibition of protein activity

[0224] In some embodiments of the invention, the polynucleotide encodes an antibody that binds to at least one polypeptide encoded by the S gene and reduces the activity of that polypeptide. In another embodiment, antibody binding leads to increased turnover of the antibody-peptide complex via a cell quality control mechanism. Antibody expression in plant cells and molecular pathways through which expression is inhibited, as well as antibody binding to proteins in plant cells, are known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21: 35-36.

[0225] 4. Gene damage

[0226] In some embodiments of the present invention, the activity of the polypeptide encoded by the S gene can be reduced or eliminated by disrupting the S gene encoding the polypeptide. The S gene encoding the polypeptide can be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenesis of the plant using random or directed mutagenesis, and plants with the desired trait are selected.

[0227] i. Transposable sublabels

[0228] In one embodiment of the invention, a transposon tag is used to reduce or eliminate the activity of one or more polypeptides encoded by the S gene. The transposon tag includes inserting a transposon into the endogenous S gene to reduce or eliminate polypeptide expression.

[0229] In this embodiment, the expression of more than one polypeptide encoded by the S gene is reduced or eliminated by inserting transposons into the regulatory or coding regions of the S gene encoding the polypeptide. Transposons located in exons, introns, 5' or 3' untranslated sequences, promoters, or other regulatory sequences of the S gene can be used to reduce or eliminate the expression and / or activity of the encoded polypeptide.

[0230] Plant-specific transposon tagging methods are well known in this art. See, for example, Maes et al., (1999) Trends Plant ScL 4: 90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett. 179: 53-59; Meissner et al., (2000) Plant J. 22: 265-274; Phogat et al., (2000) J. Biosci. 25: 57-63; Walbot, (2000) Curr. Opin. Plant Biol. 2: 103-107; Gai et al., (2000) Nucleic Acids Res. 28: 94-96; Fitzmaurice et al., (1999) Genetics 153: 1919-1928. In addition, the TUSC procedure for screening Mu insertions into selected genes has been described in Bensen et al., (1995) Plant Ce / / 7:75-84; Mena et al., (1996) Science 274:1537-1540; and U.S. Patent No. 5,962,764.

[0231] ii. Mutant plants with reduced activity

[0232] Other methods for reducing or eliminating endogenous gene expression in plants are also known in the art and can be similarly applied to this invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis using reverse genetics techniques (and PCR) to identify plant lines with endogenous gene deletions, and fast neutron deletion mutagenesis. For example, see Ohshima et al., (1998) Virology 243: 472-481; Okubara et al., (1994) Genetics 137: 867-874; and Quesada et al., (2000) Genetics 154: 421-436. Furthermore, the rapid and automated method of TILLING (directed induction of local genomic mutations) for screening chemically induced mutations can also be applied to this invention, which uses selective endonuclease digestion of denaturing HPLC or PCR products. See McCallum et al., (2000) Nat. Biotechnol. 18: 455-457.

[0233] Mutations that affect gene expression or interfere with the function of encoded proteins are known in this art. Insertion mutations in gene exons typically result in null mutants. Mutations on conserved residues are particularly effective in suppressing the activity of encoded proteins. Conserved residues of peptides suitable for mutagenesis to eliminate activity have been described. These mutants can be isolated according to well-known procedures, and mutations at different sites can be superimposed through genetic hybridization. See, for example, Gruis et al., (2002) PlantCell 14:2863-2882.

[0234] In another embodiment of the invention, a dominant mutant can be used to induce RNA silencing due to recombination at gene inversion and duplication sites. See, for example, Kusaba, et al., (2003) Plant Cell 15: 1455-1467.

[0235] This invention includes other methods for altering (including reducing, inducing, or eliminating) the activity of one or more polypeptides encoded by the S gene. Examples of other methods for altering or mutating the nucleotide sequence of a plant genome are known in the art and include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutant vectors, RNA:DNA repair vectors, mixed double-stranded oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinant oligonucleotide bases. These vectors and methods of use are known in the art. See, for example, U.S. Patents 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984. See also WO 98 / 49350, WO 99 / 07865, WO 99 / 25821, and Beetham et al., (1999) Proc. Natl. Acad. Sci. USA 96: 8774-8778.

[0236] Genome editing and induced mutations

[0237] Generally, methods for modifying or altering the host's endogenous genomic DNA are available. This includes altering the sequence of the host's endogenous DNA or transgenic sequences with pre-existing regulatory elements, including regulatory elements, coding and non-coding sequences. These methods can also be used to target nucleic acids to pre-modified target recognition sequences in the genome. For example, the genetically modified cells or plants described herein are produced using a wide range of nucleases "custom-made" for modifying the plant genome (see, for example, WO 2009 / 114321; Gao et al., (2010) Plant Journal 1: 176-187). Other targeted modifications are achieved by using zinc finger domain recognition plus restriction enzymes. See, for example, Urnov et al., (2010) Nat Rev Genet. 11(9): 636-46; Shukla et al., (2009) Nature 459(7245): 437-41.

[0238] “TILLING” or “directed induction of local genomic mutation” refers to mutagenesis techniques that can be used to generate and / or identify and ultimately isolate mutagenized variants of specific nucleic acids that regulate expression and / or activity (McCallum et al., (2000), Plant Physiology 123: 439-442; McCallum et al., (2000) Nature Biotechnology 18: 455-457; and Colbert et al., (2001) Plant Physiology 126: 480-484).

[0239] TILLING combines high-density point mutations with rapid and sensitive mutation detection. Typically, ethyl methanesulfonate (EMS) is used to induce mutagenesis in plant seeds. EMS alkylates guanine, which often leads to mismatches. For example, seeds are soaked in an EMS solution of approximately 10–20 mM for 10–20 hours; the seeds are then washed and sown. This generation of plants is called M1. M1 plants are then self-fertilized. Mutations present in the cells forming reproductive tissues are inherited through the next generation (M2). Typically, M2 plants are screened for mutations in desired genes and / or for specific phenotypes.

[0240] TILLING also allows for the selection of plants carrying mutants. These mutants can exhibit altered expression in terms of intensity, location, or timing (e.g., if the mutation affects the promoter). These mutants can exhibit activity higher or lower than that expressed by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei and Koncz, (1992) In Methods in Arabidopsis Research, edited by Koncz et al., Singapore, World Scientific Publishing Co., pp. 16-82; Feldmann et al., (1994) In Arabidopsis. Edited by Meyerowitz and Somerville, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 137-172; Lightner and Caspar, (1998) In Methods on Molecular Biology 82:91-104; Edited by Martinez Zapater and Salinas, Humana Press, Totowa, NJ.); (b) DNA preparation and mixing of individuals; (c) PCR amplification of the region of interest; (d) denaturation and annealing to form heteroduplexes; (e) DHPLC to detect the presence of heteroduplexes in the sample pool as additional peaks in the chromatography; (f) identification of mutant individuals; and (g) sequencing of the mutant PCR products. The TILLING method is known in the art (US Patent No. 8,071,840).

[0241] Other mutagenesis methods may also be used to introduce mutations into the disclosed genes. Methods for introducing genetic mutations into plant genes and screening for plants with the desired traits are well known. For example, seeds or other plant material may be treated with mutagenic chemicals according to standard techniques. Such chemicals include, but are not limited to, diethyl sulfate, ethyleneimine, and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from sources such as X-rays or gamma rays may be used.

[0242] Another genome editing method that can be used according to various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example, in U.S. Patent 8,697,359 and the references cited therein. In short, CRISPR is a microbial nuclease system involved in the defense against invading bacteriophages and plasmids. CRISPR sites in microbial hosts contain a combination of CRISPR-associated (Cas) genes and specific non-coding RNA elements (sgRNAs) capable of programming CRISPR-mediated nucleic acid cleavage. Three (1-111) CRISPR systems have been identified in a wide range of bacterial hosts. A key feature of each CRISPR site is the presence of a series of repetitive sequences (direct repeats) separated by short, non-repetitive sequences (spacers). A non-coding CRISPR array transcribes and cuts within the direct repeats into short crRNAs containing individual spacer sequences, guiding the Cas nuclease to the target site (the protospacer). Type II CRISPR is one of the most well-described systems and involves targeted DNA double-strand breaks in a four-step sequence. First, two non-coding RNAs (precursor crRNA array and tracrRNA) are transcribed from a CRISPR site. Second, tracrRNA hybridizes with the repeat region of the precursor crRNA and mediates the processing of the precursor crRNA into mature crRNA containing a separate spacer sequence. Third, the mature crRNA: the tracrRNA complex guides Cas9 to the target DNA via Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA adjacent to the protospacer's neighboring motif (PAM, additional requirement for target recognition). Finally, Cas9 mediates the cleavage of the target DNA to create a double-strand break within the protospacer.

[0243] Therefore, Cas9 is a characteristic protein of the type II CRISPR-Cas system, and the large monomeric DNA ribozyme is guided to a target DNA sequence adjacent to the PAM (protospacer adjacent motif) sequence via a complex of two non-coding RNAs (CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA)). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, while the RuvC-like domain cleaves the non-complementary strand, thereby introducing blunt-end cleavage in the target DNA. Heterologous expression of Cas9 with sgRNA can introduce site-specific double-strand breaks (DSBs) into the genomic DNA of living cells in various organisms. For applications in eukaryotes, a codon-optimized form of Cas9 derived from *Streptococcus pyogene* has been used.

[0244] Single-stranded guide RNA (sgRNA) is the second component of the CRISPR / Cas system, forming a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA and tracrRNA. The sgRNA guide sequence at its 5' end imparts DNA target specificity. Therefore, by modifying the guide sequence, sgRNAs with different target specificities can be generated. The typical length of the guide sequence is 20 bp. In plants, sgRNA is expressed using plant RNA polymerase III promoters, such as U6 and U3. The Cas9 expression plasmid used in the methods of this invention can be constructed as described in this technical field. Alternatively, Cas9 (or Cas12A) can be transferred as an active protein into plant tissues.

[0245] Nucleotide constructs, expression cassettes, and vectors

[0246] The use of the term "nucleotide construct" herein is not intended to limit the embodiments to nucleotide constructs containing DNA. Those skilled in the art will recognize that nucleotide constructs composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, particularly polynucleotides and oligonucleotides, can also be used in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of this embodiment also include all complementary forms of these constructs, molecules, and sequences. Furthermore, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the present invention include all nucleotide constructs, molecules, and sequences that can be used in the plant transformation methods of this embodiment, including but not limited to those comprising deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. The nucleotide constructs, nucleic acids, and nucleotide sequences of this embodiment also include all forms of nucleotide constructs, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc.

[0247] Another embodiment involves a transformed organism, such as an organism selected from plant cells, bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. The transformed organism comprises a DNA molecule of this embodiment that can be stably integrated into the genome of the transformed organism, an expression cassette containing the DNA molecule, or a vector containing the expression cassette.

[0248] The sequences provided in this embodiment are provided as DNA constructs for expression in an organism of interest. The constructs contain 5' and 3' regulatory sequences linked to the sequences of this embodiment. As used herein, the term "operably linked" refers to a functional link between a promoter and / or regulatory sequence and a second sequence, wherein the promoter and / or regulatory sequence initiates, mediates, and / or influences transcription of the DNA sequence corresponding to the second sequence. Typically, operably linked means that the linked nucleic acid sequences are contiguous and, if necessary, link two protein-coding regions in the same reading frame. The constructs may additionally contain at least one other gene to be co-transformed into an organism. Alternatively, one or more other genes may be provided on multiple DNA constructs.

[0249] Such a DNA construct has multiple restriction sites for inserting the polypeptide gene sequence of the present invention, thereby subjecting it to transcriptional regulation by the regulatory region. The DNA construct may also contain selective marker genes.

[0250] DNA constructs typically comprise, in the 5' to 3' direction of transcription: a transcription and translation initiation region (i.e., promoter) that functions in the host organism, the DNA sequence of this embodiment, and a transcription and translation termination region (i.e., termination region). The transcription initiation region (i.e., promoter) may be natural, analogous, foreign, or heterologous to the host organism and / or to the sequence of this embodiment. Furthermore, the promoter or regulatory sequence may be a natural or synthetic sequence. As used herein, the term "foreign" means that the promoter is not present in the natural organism in which the promoter is introduced. As used herein, the term "heterologous" refers to a sequence derived from a foreign species, or, if the sequence is derived from the same species, its natural form has been extensively modified in terms of composition and / or genetic locus through intentional human intervention. As used herein, chimeric genes comprise coding sequences operatively linked to a transcription initiation region heterologous to the coding sequence. In the case of a natural or native promoter sequence, the expression of the operatively linked sequence may differ from wild-type expression, resulting in phenotypic alterations.

[0251] In some embodiments, the DNA construct comprises an S-gene polynucleotide encoding a subunit polypeptide of the clathrin aptamer, a triangular pentapeptide repeat polypeptide, or an RPS2-like resistance polypeptide as described in this embodiment. In some embodiments, the DNA construct comprises an S-gene polynucleotide encoding a fusion protein comprising the polypeptide described in this embodiment.

[0252] In some implementations, the DNA construct may also include a transcription enhancer sequence. As used herein, an enhancer is a DNA sequence capable of promoting promoter activity and may be an intrinsic element of the promoter or a heterologous element inserted to enhance the promoter level or tissue specificity. Various enhancers may also be used, including, for example, introns that have gene expression-enhancing properties in plants (US Patent Application 2009 / 0144863), ubiquitin introns (i.e., maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), Omega enhancers or Omega prime enhancers (Gallie et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie et al., (1987) Gene 60: 217-25), CaMV 35S enhancers (see, for example, Benfey et al., (1990) EMBO J.9: 1685-96) and the enhancer of US Patent 7,803,992. The transcriptional enhancers listed above are not intended to be limiting. Any suitable transcriptional enhancer may be used in this embodiment.

[0253] The termination region may be natural to the transcription start region, natural to the operatively linked DNA sequence of interest, natural to the plant host, or may be of another origin (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host, or any combination thereof).

[0254] Convenient termination regions, such as those for octopaline synthase and carmine synthase, can be obtained from *Agrobacterium tumefaciens*. See also Guerineau et al., (1991) Mol Gen. Genet. 262: 141-144; Proudfoot, (1991) Cell 64: 671-674; Sanfacon et al., (1991) Genes Dev. 5: 141-149; Mogen et al., (1990) Plant Cell 2: 1261-1272; Munroe et al., (1990) Gene 91: 151-158; Balias et al., (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al., (1987) Nucleic Acid Res. 15: 9627-9639.

[0255] Where appropriate, nucleic acids can be optimized to improve expression in the host organism. Therefore, in the case of a plant as the host organism, the synthesized nucleic acid can utilize plant-preferred codons to improve expression. For an exploration of host-preferred usage, see, for example, Campbell and Gown, (1990) Plant Physiol. 92: 1-11. For example, although the nucleic acid sequence of this embodiment can be expressed in both monocot and dicot species, the sequence can be modified for specific preferences and GC content preferences of monocots or dicots, as these preferences have been shown to be different (Murray et al., (1989) Nucleic Acids Res. 17: 477-498). Thus, a plant's preference for a specific amino acid can be derived from known gene sequences in the plant.

[0256] Other sequence modifications are known to enhance gene expression in the cell host. These include eliminating sequences encoding spurious polyadenylation signals, exon-intron splicing site signals, transposon-like repeats, and other well-described sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to the average level of a given cell host calculated based on known genes expressed in a reference host cell. As used herein, the term “host cell” refers to a cell that contains the vector and is intended to support the replication and / or expression of the expression vector. Host cells can be prokaryotic cells, such as *E. coli*, or eukaryotic cells, such as yeast, insect, amphibian, or mammalian cells, or monocotyledonous or dicotyledonous plant cells. An example of a dicotyledonous host cell is a spinach host cell. Where feasible, sequences are modified to avoid predicted hairpin secondary mRNA structures.

[0257] In preparing expression cassettes, various DNA fragments can be manipulated to provide the DNA sequence in the correct orientation and, under appropriate conditions, within the appropriate reading frame. This can be achieved by using adaptors or linkers to ligate the DNA fragments, or by employing other manipulations to provide convenient restriction sites, remove redundant DNA, and eliminate restriction sites. This can involve in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, and substitution, such as transitions and transversions.

[0258] A variety of promoters can be used in the practice of this embodiment. Promoters can be selected based on the desired results. Nucleic acids can be combined with constitutive promoters, tissue-biased promoters, inducible promoters, or other promoters for expression in a host organism.

[0259] Plant transformation

[0260] The method of this embodiment relates to introducing polypeptides or polynucleotides into plants. As used herein, "introduction" means the delivery of a polynucleotide or polypeptide into a plant in a manner in which its sequence can enter the interior of the plant cell. The method of this embodiment is not dependent on a specific method of introducing polynucleotides or polypeptides into a plant, as long as it allows (one or more) nucleotides or polypeptides to enter at least one cell of the plant. Methods for introducing (one or more) nucleotides or polypeptides into a plant include, but are not limited to, stable transformation, transient transformation, and virus-mediated transformation.

[0261] As used in this article, "stable transformation" refers to the integration of a nucleotide construct introduced into a plant into the plant's genome and its ability to be inherited by its offspring. As used in this article, "transient transformation" refers to the introduction of a polynucleotide into a plant that does not integrate into the plant's genome, or the introduction of a polypeptide into a plant.

[0262] Transformation schemes and schemes for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell used for transformation (i.e., monocots or dicots). Appropriate methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome include microinjection (Crossway et al., (1986) Biotechniques 4: 320-334), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606), Agrobacterium-mediated transformation (US Patents 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al., (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, US Patents 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe et al., (1988) Biotechnology 6: 923-926) and Led transformation (WO 00 / 28058), etc. For potato transformation, see Tu et al., (1998) Plant Molecular Biology 37: 829-838 and Chong et al., (2000) Transgenic Research 9: 71-78. Other transformation processes can be found in Weissinger et al., (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al., (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al., (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al., (1988) Bio / Technology 6: 923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al., (1998) Theor. AppL Genet. 96: 319-324 (soybeans); Datta, et al., (1990) Biotechnology 8: 736-740 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci.USA 85: 4305-4309 (maize); Klein et al., (1988) Biotechnology 6: 559-563 (maize); US Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol. 91: 440-444 (maize); Fromm et al., (1990) Biotechnology 8: 833-839 (maize); Hooykaas-Van Slogteren et al., (1984) Nature (London) 311: 763-764; US Patent No. 5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al., (1992) Theor. AppL Genet. 84: 560-566 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al., (1993) Plant Cell Reports 12: 250-255 and Christou and Ford, (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al., (1996) Nature Biotechnology 14:745-750 (maize, via Agrobacterium tumefaciens).

[0263] Methods of introducing genome editing technology into plants

[0264] In some implementations, genome editing techniques can be used to introduce polynucleotide compositions into the plant genome, or to edit endogenous polynucleotides in the plant genome. For example, identified polynucleotides can be introduced into desired locations in the plant genome using double-strand break techniques, such as TALEN, large-scale nucleases, zinc finger nucleases, CRISPR-Cas, etc.

[0265] In some embodiments, where the S gene has already been identified in the genome, genome editing techniques can be used to alter or modify the polynucleotide sequence. Site-specific modifications that can introduce the desired allelic polynucleotide include those produced using any method that introduces site-specific modifications, including but not limited to those using gene repair oligonucleotides (e.g., U.S. Patent Application 2013 / 0019349) or using double-strand break techniques such as TALENs, large-scale nucleases, zinc finger nucleases, CRISPR-Cas, etc. Such techniques can be used to modify polynucleotides by inserting, deleting, or replacing nucleotides within the polynucleotide. Alternatively, double-strand break techniques can be used to add other nucleotide sequences to the polynucleotide. Other sequences that can be added include other expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing techniques can be used to locate other disease resistance proteins closely adjacent to the S gene polynucleotide within the plant genome to generate a molecular stack of disease resistance proteins. The terms “altered target site,” “altered target sequence,” “modified target site,” and “modified target sequence” are used interchangeably herein and, as disclosed herein, refer to a target sequence containing at least one altered target sequence when compared with a non-altered target sequence. Such “alteration” includes, for example: (i) substitution of at least one nucleotide (substitution, e.g., GC to AT, or e.g., G transpose to C or A transpose to T), (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0266] Implementation

[0267] The following numbered embodiments also form part of this invention:

[0268] 1. A method for reducing plant susceptibility to pathogens, the method comprising: introducing one or more nucleotide modifications at a locus in the plant, wherein the locus contains a susceptibility gene (S gene) encoding a subunit protein of a clathrin aptamer, a family protein containing a triangular pentapeptide repeat sequence, or an RPS2-like resistance protein.

[0269] 2. The method as described in Embodiment 1, wherein the one or more nucleotide modifications include multiple transitions such as GC to AT, transversions such as G to C or A to T, insertions, deletions or combinations thereof in the S gene.

[0270] 3. The method as described in Embodiment 1 or 2, wherein the one or more nucleotide modifications are introduced by a mutagenic factor.

[0271] 4. The method as described in Embodiment 3, wherein the mutagenic factor is ethyl methanesulfonate (EMS).

[0272] 5. The method as described in Embodiment 1 or 2, wherein the one or more nucleotide modifications are introduced via targeted DNA modification.

[0273] 6. The method as described in Embodiment 5, wherein the targeted DNA modification is introduced via an RNA-guided endonuclease.

[0274] 7. The method as described in embodiment 1 or 2, wherein the one or more nucleotide modifications are introduced by infiltration.

[0275] 8. The method of any one of embodiments 1-7, wherein the one or more nucleotide modifications result in changes in the expression or activity of the protein encoded by the S gene.

[0276] 9. The method of any one of embodiments 1-8, wherein the one or more nucleotide modifications include insertions, deletions or single nucleotide polymorphisms (SNPs).

[0277] 10. The method of any one of embodiments 1-9, wherein the more than one nucleotide modification includes at least one non-synonymous single nucleotide polymorphism (nsSNP) in the coding sequence of the S gene.

[0278] 11. The method of any one of embodiments 1-10, wherein the one or more nucleotide modifications are present in the (a) coding region, (b) non-coding region, (c) regulatory sequence, or (d) untranslated region of the S gene.

[0279] 12. The method of any one of embodiments 1-11, wherein the one or more nucleotide modifications result in one or more of the following: (a) alteration of S gene expression; (b) generation of one or more alternative splicing transcripts of the S gene; (c) deletion or alteration of one or more DNA-binding domains; (d) frameshift mutation in one or more exons of the S gene; (e) substantial deletion or alteration of the S gene, or deletion of the full-length open reading frame of the S gene; (f) inhibition or induction of enhancer motifs present in the regulatory region encoding the S gene; (g) modification or deletion of one or more nucleotides of a regulatory element operatively linked to the expression of the S gene, wherein the regulatory element is present in a promoter, intron, 3'UTR, terminator, or combination thereof.

[0280] 13. The method of any one of embodiments 1-12, wherein the more than one nucleotide modification produces a sequence selected from SEQ ID NOs: 1, 2, 93, 94, 169 or 170.

[0281] 14. A method for reducing plant susceptibility to pathogens, the method comprising: altering the activity or expression of a protein encoded by a susceptibility gene (S gene), wherein the protein is a clathrin aptamer subunit protein, a family protein containing a triangular pentapeptide repeat sequence, or an RPS2-like resistance protein.

[0282] 15. The method of embodiment 14, wherein the alteration includes one or more mechanisms selected from RNA interference, genome editing including gene substitution or allele substitution, gene knockout, and gene knockdown.

[0283] 16. The method of any one of embodiments 1-15, wherein the S gene encodes a clathrin aptamer subunit protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NOs: 6, 64-92, 294-315.

[0284] 17. The method of embodiment 16, wherein the S gene encodes a clathrin aptamer subunit protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NO: 6.

[0285] 18. The method of any one of embodiments 1-15, wherein the S gene encodes a triangular pentapeptide repeat protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NOs: 98, 145-168, 326-330.

[0286] 19. The method of embodiment 18, wherein the S gene encodes a triangular pentapeptide repeat protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NO. 98.

[0287] 20. The method of any one of embodiments 1-15, wherein the S gene encodes an RPS2-like resistance protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NOs: 174, 219-240, 333.

[0288] 21. The method of any one of Embodiment 20, wherein the S gene encodes an RPS2-like resistance protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NO: 174.

[0289] 22. The method of any one of embodiments 1-21, wherein the locus comprises and is flanked by SEQ ID NO: 241 and SEQ ID NO: 251.

[0290] 23. The method of any one of embodiments 1-22, wherein the plant is a species of the genus Spinacia oleracea, and the pathogen belongs to the phylum Oomycota.

[0291] 24. The method of any one of embodiments 23, wherein the pathogen is selected from *Peronospora farinosa* (downy mildew); *Peronospora effusa* (downy mildew); *Stemphylium botryosum f.sp.spinacia* (leaf spot); *Cladosporium variabile* (leaf spot); *Albugo occidentals* (white rust); *Colletotrichum dematium f.sp.spinaciae* (anthracnose); *Pythium* species (damping-off / seedling blight); *Fusarium oxysporum f.sp.spinaciae* (wilt); *Aphanomyces cochlioides* (black root rot); and *Verticillium*. At least one species of dahlia (chlorosis).

[0292] 25. The method of any one of embodiments 1-22, wherein the plant is a sunflower (Helianthus) and the pathogen is a species of downy mildew (Plasmopara halstedii).

[0293] 26. The method of any one of embodiments 1-22, wherein the plant is a species of maize (Zea mays) and the pathogen is a species of maize downy mildew (Peronosclerospora maydis).

[0294] 27. The method of any one of embodiments 1-22, wherein the plant is a beet (Betavulgaris) species and the pathogen is a downy mildew (Peronospora farinosa) species (downy mildew).

[0295] 28. The method of any one of embodiments 1-22, wherein the plant is a species of lettuce (Lactucasativa) and the pathogen is a species of Bremia lactucae (downy mildew).

[0296] 29. The method of any one of embodiments 1-22, wherein the plant belongs to the Cucurbitaceae family and the pathogen is the species *Pseudoperonospora cubensis* (downy mildew).

[0297] 30. The method of any one of embodiments 1-22, wherein the plant belongs to the Brassicaceae family and the pathogen is a species of parasitic downy mildew (Peronospora parasitica).

[0298] 31. The method of any one of embodiments 1-22, wherein the plant belongs to the Solanaceae family and the pathogen is the species Peronospora hyoscyami (downy mildew).

[0299] 32. A plant with reduced susceptibility to pathogens, comprising one or more nucleotide modifications at a locus in the plant, wherein the locus comprises a susceptibility gene (S gene) encoding a subunit protein of a clathrin aptamer, a family protein containing a triangular pentapeptide repeat sequence, or an RPS2-like resistance protein.

[0300] 33. The plant as described in embodiment 32, wherein the one or more nucleotide modifications include multiple transitions such as GC to AT, transversions such as G to C or A to T, insertions, deletions or combinations thereof in the S gene.

[0301] 34. The plant as described in embodiment 32 or 33, wherein the one or more nucleotide modifications result in alteration of the expression or activity of the protein encoded by the S gene.

[0302] 35. The plant as described in any one of embodiments 32-34, wherein the more than one nucleotide modification includes insertion, deletion or single nucleotide polymorphism (SNP).

[0303] 36. The plant as described in any one of embodiments 32-35, wherein the one or more nucleotide modifications are present in the (a) coding region, (b) non-coding region, (c) regulatory sequence, or (d) untranslated region of the S gene.

[0304] 37. The plant as described in any one of embodiments 32-36, wherein the one or more nucleotide modifications include at least one non-synonymous single nucleotide polymorphism (nsSNP) in the coding sequence of the S gene.

[0305] 38. The plant according to any one of embodiments 32-37, wherein the one or more nucleotide modifications result in one or more of the following: (a) alteration of S gene expression; (b) production of one or more alternative splice transcripts of the S gene; (c) deletion or alteration of one or more DNA-binding domains; (d) frameshift mutation in one or more exons of the S gene; (e) substantial deletion or alteration of the S gene, or deletion of the full-length open reading frame of the S gene; (f) repression or induction of enhancer motifs present in the regulatory region encoding the S gene; (g) modification or deletion of one or more nucleotides of a regulatory element operatively linked to the expression of the S gene, wherein the regulatory element is present in a promoter, intron, 3'UTR, terminator, or combination thereof.

[0306] 39. The plant as described in any one of embodiments 32-38, wherein the one or more nucleotide modifications produce a sequence selected from SEQ ID NOs: 1, 2, 93, 94, 169 or 170.

[0307] 40. A plant with reduced susceptibility to pathogens, comprising altered susceptibility gene (S gene) encoding protein activity or expression, wherein said protein is a clathrin aptamer subunit protein, a family protein containing a triangular pentapeptide repeat sequence, or an RPS2-like resistance protein.

[0308] 41. The plant as described in any one of embodiments 32-40, wherein the S gene encodes a clathrin aptamer subunit protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NOs: 6, 64-92, 294-315.

[0309] 42. The plant of any one of embodiments 32-40, wherein the S gene encodes a trigonal pentapeptide repeat protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NOs: 98, 145-168, 326-330.

[0310] 43. The plant of any one of embodiments 32-40, wherein the S gene encodes an RPS2-like resistance protein comprising an amino acid sequence having at least 80% identity with a sequence selected from SEQ ID NOs: 174, 219-240, 333.

[0311] 44. The plant as described in any one of embodiments 32-43, wherein the locus comprises and is flanked by SEQ ID NO: 241 and SEQ ID NO: 251.

[0312] 45. The plant as described in any one of embodiments 32-44, wherein the plant is a species of the genus Spinacia oleracea, and the pathogen belongs to the phylum Oomycota.

[0313] 46. ​​The plant as described in embodiment 45, wherein the pathogen is selected from *Peronospora farinosa* (downy mildew); *Peronospora effusa* (downy mildew); *Stemphylium botryosum f.sp.spinacia* (spinacia leaf spot); *Cladosporium variabile* (cladosporium leaf spot); *Albugo occidentals* (white rust); *Colletotrichum dematium f.sp.spinaciae* (anthracnose); *Pythium* species (damping-off / seedling blight); *Fusarium oxysporum f.sp.spinaciae* (wilt); *Aphanomyces cochlioides* (black root rot); and *Verticillium*. At least one species of dahlia (chlorosis).

[0314] 47. The plant as described in any one of embodiments 32-44, wherein the plant is a sunflower (Helianthus) and the pathogen is a species of Plasmopara halstedii (downy mildew).

[0315] 48. The plant as described in any one of embodiments 32-44, wherein the plant is a species of maize (Zea mays) and the pathogen is a species of maize downy mildew (Peronosclerospora maydis).

[0316] 49. The plant as described in any one of embodiments 32-44, wherein the plant is a beet (Betavulgaris) species and the pathogen is a downy mildew (Peronospora farinosa) species (downy mildew).

[0317] 50. The plant as described in any one of embodiments 32-44, wherein the plant is a species of lettuce (Lactucasativa) and the pathogen is a species of Bremia lactucae (downy mildew).

[0318] 51. The plant as described in any one of embodiments 32-44, wherein the plant belongs to the Cucurbitaceae family, and the pathogen is a species of Pseudoperonospora cubensis (downy mildew).

[0319] 52. The plant as described in any one of embodiments 32-44, wherein the plant belongs to the Brassicaceae family, and the pathogen is a species of parasitic downy mildew (Peronospora parasitica).

[0320] 53. The plant as described in any one of embodiments 32-44, wherein the plant belongs to the Solanaceae family and the pathogen is the species Peronospora hyoscyami (downy mildew).

[0321] 54. A method for identifying spinach plants with enhanced resistance to downy mildew, the method comprising: a) detecting in spinach plants alleles associated with enhanced resistance to downy mildew, wherein the resistance alleles contain “T” in Spov3_chr4_93275081, “A” in Spov3_chr4_105049870, “A” in Spov3_chr4_107241402, “A” in Spov3_chr4_109546568, and “A” in Spov3_chr4_109698808. The following are possible interpretations of Spov3_chr4_112008390, Spov3_chr4_112574123, Spov3_chr4_117783935, Spov3_chr4_118191085, Spov3_chr4_118788268, or Spov3_chr4_121142541: "A", "T", "T", "T", "A ...

[0322] 55. A method for identifying and / or selecting spinach plants with enhanced resistance to downy mildew, the method comprising: a) screening a population having a marker located on chromosome 4 containing and flanked by SEQ ID NO: 241 and SEQ ID NO: 251 to determine whether there is more than one plant containing a resistance allele in the population; and b) selecting at least one plant containing the resistance allele from the population.

[0323] 56. The method of embodiment 55 further includes: c) crossing the plant of b) with a second plant; and d) obtaining offspring plants with resistance alleles.

[0324] 57. The method as described in embodiment 55 or 56, wherein the resistance allele contains "T" in Spov3_chr4_93275081, "A" in Spov3_chr4_105049870, "A" in Spov3_chr4_107241402, "A" in Spov3_chr4_109546568, and "A" in Spov3_chr4_109698808. The following conditions must be met: Spov3_chr4_112008390 contains “A”; Spov3_chr4_112574123 contains “T”; Spov3_chr4_117783935 contains “T”; Spov3_chr4_118191085 contains “A”; Spov3_chr4_118788268 contains “A”; or Spov3_chr4_121142541 contains “A”.

[0325] 58. A spinach plant comprising broad-spectrum resistance to spreading downy mildew, wherein said resistance is conferred by an introgression fragment comprising a susceptibility gene (S gene).

[0326] 59. The spinach plant as described in embodiment 58, wherein the S gene is present in plants cultured from seeds of Nr.22-18, a representative seed sample deposited with accession number NCIMB 43773.

[0327] 60. The spinach plant as described in embodiment 58 or 59, wherein the infiltrated fragment comprises one or more of SEQ ID NOs: 1, 93, 169.

[0328] 61. A spinach plant as described in any one of embodiments 58-60, wherein the spinach plant contains a letter selected from "T" at Spov3_chr4_93275081, "A" at Spov3_chr4_105049870, "A" at Spov3_chr4_107241402, "A" at Spov3_chr4_109546568, and "A" at Spov3_chr4_109698808. At least one allele of the following: “A” at Spov3_chr4_112008390, “T” at Spov3_chr4_112574123, “T” at Spov3_chr4_117783935, “A” at Spov3_chr4_118191085, “A” at Spov3_chr4_118788268, and “A” at Spov3_chr4_121142541.

[0329] 62. Spinach plants as described in any one of embodiments 58-61, wherein the spinach plants contain at least resistance to spread downy mildew races 1-20, including but not limited to Pe:4+, Pe:5, Pe:12, Pe:14, Pe:16, Pe:17 and Pe:19, or at least resistance to spread downy mildew races 1-19, including but not limited to Pe:4+, Pe:5, Pe:12, Pe:14, Pe:16, Pe:17 and Pe:19, and unclassified Pe isolates 4US and UA2016-21A (Pe:21A).

[0330] 63. The spinach plant as described in any one of embodiments 58-62, wherein the spinach plant is a hybrid plant.

[0331] 64. The spinach plant as described in any one of embodiments 58-62, wherein the spinach plant is an inbred plant.

[0332] 65. Seeds of spinach plants as described in any one of embodiments 58-64 can be cultivated.

[0333] 66. Leaves of a spinach plant as described in any one of embodiments 58-64.

[0334] 67. Progeny plants of the spinach plant described in any one of embodiments 58-64.

[0335] 68. A portion of a spinach plant according to any one of embodiments 58-64, wherein the portion is selected from stems, branches, petioles, cotyledons, flowers, anthers, pollen, ovaries, roots, root tips, protoplasts, callus, microspores, stalks, ovules, branches, seeds, embryos, embryo sacs, cells, meristems, buds, or leaves.

[0336] 69. A cell culture or tissue culture comprising cells or tissues derived from the portion described in embodiment 68.

[0337] 70. A food product comprising harvested leaves of a spinach plant as described in any one of embodiments 58-64.

[0338] 71. A container comprising a spinach plant as described in any one of embodiments 58-64, the spinach plant being situated in a growth substrate for harvesting leaves from the plant.

[0339] All publications and patent applications mentioned in this specification represent the skill of a person skilled in the art to which this patent pertains. All publications and patent applications are incorporated herein by reference to the same extent as each individual publication or patent application expressly and individually indicates its incorporation by reference.

[0340] Although the invention has been described in detail by way of illustration and examples for the purpose of clarity, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

[0341] The following examples are provided in an explanatory rather than restrictive manner.

[0342] Example

[0343] Note: Although Examples 1 to 7 below focus primarily on and show the results of experiments conducted with the propagule race Pe:16, very similar results were obtained with propagule races Pe:4+, Pe:5, Pe:12, Pe:14, Pe:17, Pe:19 and Pe:21A.

[0344] Example 1: Identification of EMS-induced variants resistant to spreading downy mildew

[0345] The spinach inbred line B11-509-7-41-2sel. (hereinafter referred to as line B11-509) is resistant to races Pe:1 to Pe:15, Pe:17, and Pe:18, but susceptible to Pe:16, Pe:19, and other unclassified Pe isolates, such as 4US and UA2016-21A. Seeds of line B11-509 were treated with 0.4% ethyl methanesulfonate (EMS) to induce genome-wide GC>AT transitions. EMS also induced other changes at low frequencies. DM resistance was screened by self-pollination of individual M1 plants and by inoculating seedlings of the M2 family with isolate Pe:16. EMS22 of the M2 family was found to separate into DM-resistant and susceptible plants. The M3 family was obtained by self-pollination of 3 resistant M2 plants and 7 susceptible M2 plants. The resistance and susceptibility patterns within the 10 M3 families were confirmed by testing with isolate Pe:16 again. Testing with the unclassified isolate Pe:21A showed that the resistance is more broadly effective and, due to the mechanism of action of this resistance, may be effective against all other existing and future races of Spreading Downy mildew.

[0346] Two approaches were used to investigate which chromosomal region and ultimately which specific mutation was causally related to the observed DM resistance: a mixed grouping analysis (BSA) of self-crossed M3 families and genetic mapping in the Viroflay backcross F2 population.

[0347] BSA was performed on mixed gDNA from 3 resistant M3 families and 7 susceptible M3 families (R pool and S pool). Both pools were resequencing and plotted on the Viroflay reference genome Phytozome: Soleracea_575_Spov3, yielding (1) differences between (2) and (3) pools of the B11-509 strain used for EMS treatment. Scanning plots of SNP coverage on the 6 chromosomes in both pools showed differences in the 20 Mb adjacent region and the 6.2 Mb distal region of Chr4. Figure 1 The highest percentage of near-homozygous R pool SNPs and a very low percentage of S pool SNPs were detected, and none were present in the B11-509 data. SNPs in these regions validated the effectiveness of the gene model for the reference genome: some SNPs were located in exons of predicted genes, and three SNPs resulted in nonsynonymous amino acid alterations. These three nonsynonymous alterations are predicted to affect the genes Spov3_chr4.04649 (the putative resistance RPS2-like protein), Spov3_chr4.04219 (associated with the putative family of proteins containing triangular pentapeptide repeat sequences), and Spov3_chr4.04752 (associated with the putative subunit protein in the clathrin aptamer).

[0348] Note: The gene model Spov3_chr4.04129 is incorrect and should be replaced with the Sp76 gene model Spo10764 (SEQ ID NO: 96, see Table 3).

[0349] For genetic mapping, three resistant M3 plants were backcrossed with Viroflay (susceptible to all Pe races). Phenotypic analysis of the segregating F2 plants from each hybrid was performed using Pe:16, and genotyping was conducted using the KASP test with 142 R pool-specific SNPs covering all six chromosomes from BSA. Phenotypic analysis of approximately 2000 F2 plants from 11 F2 progeny generations revealed a significant 1R:3S ratio, consistent with the expected recessive impaired S gene inheritance. KASP genotyping yielded 91 informative segregating SNPs, and all positive and negative control scores were consistent. GWAS analysis of the 91 SNPs providing EMS transition and susceptibility / resistance scores to Pe:16 indicated a clear association with 11 distal Chr4 SNPs, as in BSA. Figure 2 This includes three SNPs derived from BSA from three candidate S genes.

[0350] Example 2: Identification of the causal gene (S gene) for resistance

[0351] Note: In this context, the S gene or susceptibility gene can also be referred to as the causal gene of resistance. In this context, "resistance" is the loss of susceptibility.

[0352] A clear correlation was detected between the susceptibility site (S site) and the 11 SNPs located distal to Chr4 (see Example 1 and...). Figure 2The region spans 6.2 Mb. To further refine the localization of the S locus and address its stability in other spinach backgrounds, two new hybridizations were performed: a specific EMS22 inbred line (X20-10-1) was crossed with Viroflay, and the EMS22 inbred line was crossed with a PV backbone line containing three DM resistance loci, termed RPF13 (X17-003). Phenotypic identification of each F2 plantlet from the hybridization was performed using Pe:16, and genotyping was determined using the KASP test with 11 SNPs. Phenotypic identification of several thousand F3 plants from both backgrounds revealed a significant 1R:3S ratio, consistent with the expectation of recessive damaged S gene inheritance. No significant phenotypic differences were detected between the two backgrounds. Genotyping results indicated that the potential mutation leading to resistance to Pe:16 was located near SNP 117783935_GA (SEQ ID NO: 337) in the candidate gene Spov3_chr4.04649 (the putative resistance RPS2-like protein). For further fine mapping, 70 B11-509 / Viroflay-specific SNPs were selected from genomic regions 112-118 Mb and used to detect recombination in two backgrounds. Three SNPs with the highest association were found in the region of the RPS2-like candidate gene. Figure 3 In one population from two backgrounds, the most closely associated recombination events with the RPS2-like gene were detected, yielding a 0.62 Mb region located between two flanking SNPs, SNP 117443435_AG (SEQ ID NO: 336) and >118064634^TC (SEQ ID NO: 338) (sequences listed below). Further investigation focused on the RPS2-like gene.

[0353] SNP list

[0354] >i17443435_AG-SEQ ID NO: 336

[0355]

[0356] >117783935_GA-SEQ ID NO: 337

[0357]

[0358] >118064634_TC-SEQ ID NO: 338

[0359]

[0360] Example 3: Identification of causal mutations related to plant resistance to spreading downy mildew

[0361] In addition to the reference genome Viroflay Spov3 used in the aforementioned BSA, new high-quality references for Viroflay and the inbred line B11-509 were constructed. A 0.62 Mb distal region of Chr4 containing the S site was compared using whole-genome alignment analysis. Short reads from a mutant M3 plant (EMS22-20) were obtained and used for mapping the new references. EMS is known to induce other changes at a low frequency besides genome-wide GC>AT switching. Therefore, the new read mappings were used for visual inspection of small nucleotide variations as well as larger deletions and duplications. A large deletion was detected in the gene model SOV1g044450 (an ortholog of the candidate gene Spov3_chr4.04649 (RPS2-like) in the new high-quality Viroflay reference). The deletion was close to a previously detected G>A switching leading to a change in neutral nonsynonymous amino acids. The deletion was 191 bp and confirmed in the read mappings of the resistant plant pool used in the BSA. The deletion was not present in the read mappings of the susceptible plant pools used in B11-509 and BSA. The deletion could be reconstructed by reassembling the unmapped reads of EMS22-20 in contig_59494. Three other alterations were detected in the alignment of contig_59494 with SOV1g044450: a rare G>C transition at the left boundary of the deletion, a T insertion at the left boundary of the deletion, and a G insertion at the right boundary. Figure 4 (and the sequences listed below). All changes in SOV1g044450 were confirmed by sequencing of individual PCR products from resistant EMS-induced plants and were not present in the PCR products of susceptible families and controls. Furthermore, the full-length cDNAs of EMS22-20 and B11-509 were cloned and sequenced to confirm the changes. Due to the aforementioned insertions, a deletion of 189 bp resulted in a one-amino acid change, K491N. The G>C conversion also resulted in a one-amino acid change, L493C. The G>A conversion detected in BSA resulted in a third amino acid change, G416R (…). Figure 5 (and the sequences listed below). The RPS2-like gene encodes a putative NLR-class resistance gene containing an N-terminal nucleotide-binding motif and a 5-leucine repeat (LRR) motif. A 189 bp deletion results in the removal of two C-terminal LRR motifs from the protein. In summary, the EMS-treated mutants of the EMS22 family resistant to pertussis contain causal mutations in the gene SOV1g044450: two nucleotide transitions, two nucleotide insertions, and a 189 bp deletion, resulting in a three-amino acid alteration and a 63-amino acid deletion containing two C-terminal LRRs.

[0362] Nr22-20pl5-EMSline_S3_L001_(paired)_contig_59494-RC SOV1g044450.1 Genome sequence / site - SEQ ID NO: 339

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369] SOV1g044450.1_coded sequence - SEQ ID NO: 340

[0370]

[0371]

[0372] SOV1g044450.1_protein-SEQ ID NO: 341

[0373]

[0374]

[0375] SOV1g044450-cds-missing-1-SEQ ID NO: 342

[0376]

[0377] Example 4: RNA-seq analysis of transcriptome data from the EMS22-20 mutant

[0378] To understand the resistance mechanism of the EMS22 mutant, transcriptome data were obtained through RNA-seq analysis. For this experiment, B11-509 and EMS22-20 were used as simulated controls, or a concentration of 1×10⁻⁶ was used. 5Pe:16 infection with spores / mL. Leaf samples were collected from at least 3 plants for each treatment at different time points post-infection (0, 4, 8, 24, and 48 hours post-inoculation [hpi]). RNA was isolated. A total of 20 equimolar mixtures were analyzed. FPKM values ​​of the RNA-seq data files were obtained. The RPS2-like gene (SOV1g044450) showed differential expression, with higher expression in the EMS22 mutant than in the B11-509 background. Figure 6 Visual examination of the two simulated control and treatment pools revealed identical changes in the gene's mapping data. RNA-seq results from B11-509 and EMS22-20 were used to analyze the expression levels of known defense pathways (e.g., SA, JA / Et, ABA, MAPK) in spinach orthologs. This study indicates that most of these orthologs are differentially expressed in the mutants. Therefore, this study suggests that the mutants exhibit enhanced basal resistance levels due to differential expression of multiple resistance-promoting genes (defense-related genes), such as PR genes (PR1-PR5b), chitinase, peroxidase, caffeate-O-methyltransferase, aspartate protease, and MAPK3.

[0379] Example 5: In vivo plant resistance of the EMS22-20 strain against DM isolate Pe:16

[0380] To understand the resistance mechanism of the EMS22 mutant, detailed microscopic analysis was performed, and trypan blue staining was used to track the infection process of DM isolate Pe:16 on leaves of B11-509 and EMS22-20 over time. In the infection process of B11-509, appressorium penetration and hyphal growth were visible from day 2 onwards. Furthermore, haustoria were confirmed to form. In the case of the EMS22-20 mutant, it was evident that downy mildew could not penetrate the leaf epidermis. Appressorium formed, but hyphae remained on the leaf surface, and clusters of spores and hyphae were typically observed. Figure 7 and 8 ).

[0381] Example 6: In vivo plant resistance of the EMS22-20 strain to other diseases in spinach

[0382] Diseases affecting spinach plants include, but are not limited to, anthracnose (*Colletotrichum spinosa*), damping-off / seedling blight (*Pythium cerevisiae*), downy mildew (*Pythium dispersum*), wilt (*Fusarium oxysporum*), creeping scab (*Stemphylium versicarium*, *Stemphylium beticola*, *Stemphylium drummondii*), yellowing wilt (*Verticillium dahliae*), white rust (*White rust spp.*), black root rot (*Hylocereus spp.*), and cladoceran leaf spot (*Cladosporium variegatum*). To determine whether the resistance observed in EMS22-20 is broadly effective against other spinach pathogens, several disease tests were performed. Disease symptoms were assessed on lines carrying homozygous causal mutations of the RPS2-like gene (from EMS22-20), and other lines with wild-type alleles (including untreated B11-509), ranging from 1 (very susceptible / plant death) to 9 (very resistant, asymptomatic). Table 7 shows that the RPS2-like mutant allele generated by EMS also provides enhanced resistance to *Colletotrichum gloeosporioides*, the causal agent of anthracnose in spinach, indicating that the resistance identified in EMS22-20 has broad resistance activity beyond DM. The average disease score (24 plants per line) of plants carrying the resistant RPS2-like allele was "7 or 8", compared to "below 3" for B11-509, Viroflay, and two other susceptible varieties. Interestingly, the resistance score was also higher than that of Stanton (score "6"), a spinach variety considered to have the most advanced anthracnose resistance in this field. This study demonstrates that the S allele of the EMS22-20 mutant results in broad resistance to a variety of pathogens and diseases common in spinach, leading to the assumption that similar resistance to other pathogens and / or diseases is expected.

[0383] Table 7:

[0384] Results of anthracnose testing in spinach. Twenty-four plants from each line were infected with *Colletotrichum gloeosporioides* and evaluated on a scale of 1 (very susceptible) to 9 (completely resistant). Lines carrying the mutant allele at the S locus (X20-010-1) showed high resistance compared to all other lines, including the best commercially available variety (Stanton). Homozygous mutant S loci present in each line are indicated as “Y” = present and “N” = absent. “Susceptible control” indicates a line known to be susceptible to *Colletotrichum gloeosporioides* (negative control), while “resistant control” indicates a line known to be resistant to *Colletotrichum gloeosporioides* (positive control).

[0385]

[0386] Example 7: Functional verification of RPS2-induced resistance of EMS22-20 strain to DM isolate Pe:16

[0387] Because stable transformation of spinach is not achievable, a transient expression test was established to analyze the gains and losses in resistance. The full-length coding sequences of the wild-type (WT) and mutant alleles of the candidate gene were cloned into an overexpression (OE) construct, pK7WG2, controlled by the 35S promoter. To examine expression levels in inoculated tissues, leaves and cotyledons of wild-type B11-509 and the mutant EMS22-20 were inoculated with *Agrobacterium tumefaciens* containing the OE construct. Leaves from 3- or 4-week-old plants and cotyledons from 10-day-old seedlings were used for transient expression of the candidate gene (via *Agrobacterium tumefaciens*), followed by inoculation with Pe:16 for functional validation of the candidate gene. It was hypothesized that an OE (complementation) of the RPS2-like WT allele in the EMS22-20 background would restore susceptibility of the EMS22-20 mutant to spreading downy mildew, while an OE of the mutant allele would not.

[0388] Example 8: Collinearity analysis of Spo10764 and Spov3_chr4.04752 in peas, soybeans and broad beans

[0389] Spo10764 and Spov3_chr4.04752 (Sp75 gene model ID Spo25330) are candidate genes in spinach associated with susceptibility to downy mildew.

[0390] Spo10764 was found on chromosome 1 (41, 268, 940-41, 270, 931) of the Sp75 pseudomolecularized reference chromosome (MO ID: 27091) and consists of a single exon.

[0391] Spov3_chr4.04752 was found on chromosome 1 (50, 406, 246-50, 414, 563) of the Sp75 pseudomolecularized reference chromosome (MO ID: 27091) and consists of 11 exons.

[0392] Orthologs in peas, soybeans and broad beans

[0393] The genome, CDS, and protein sequences of two candidate genes are provided. Preliminary studies of potential orthologs are conducted using a simplified BLAST approach. Subsequently, Synteny Analyser (available at galaxy-test.kws.de), which primarily utilizes JCVI and is designed for analysis of cereals, is used, but has been updated to include a wider range of crops for the purposes of this task.

[0394] Pea-Spo10764

[0395] Reference genome = cv.Caméor v1 (MO ID: 28707)

[0396] BLAST results

[0397]

[0398]

[0399] Psat0s3758g0080 was found on scaffold_03758 of the pea genome and is the best match for Spo10764 in the pea genome model.

[0400] Soybean-Spo10764

[0401] Reference genome = cv.Williams82 v4 (MO ID: 28479)

[0402] BLAST results

[0403]

[0404] Both Glyma.08G285350.1 and Glyma.08G285300.2 are located on chromosome 8 of the soybean genome and are the best match for Spo10764 in soybean gene models. Furthermore, with the default filter settings (collinear blocks consisting of at least 30 genes), Synteny Analyser also identified Glyma.08G285300.2 as an ortholog of Spo10764 in soybean cv.Williams82 v4.

[0405] Pea-Spov3_chr4.04752

[0406] Reference genome = cv.Caméor v1 (MO ID: 28707)

[0407] BLAST results

[0408]

[0409] Psat4g115080.1 and Psat2g093960.1 are located on chromosomes 2 and 4, respectively, and are the best match for Spov3_chr4.04752 in the pea gene model. Psat4g115080.1 and Psat2g093960.1 consist of 11 exons, consistent with the Spov3_chr4.04752 (Spo25330) gene model. Synteny Analyser revealed that Psat4g115080.1 is a collinear region with Spov3_chr4.04752.

[0410] Soybean-Spov3_chr4.04752

[0411] Reference genome = cv.Williams82 v4 (MO ID: 28479)

[0412] BLAST results

[0413]

[0414] Glyma.18G143100.1, Glyma.08G283400.1, Glyma.01G027300.1, and Glyma.02G037700.1 were found on chromosomes 18, 8, 1, and 2, respectively. These genes all consist of 11 exons and are consistent with the Spov3_chr4.04752 (Spo25330) gene model. Synteny Analyser identified all four gene models (Glyma.18G143100.1, Glyma.08G283400.1, Glyma.01G027300.1, and Glyma.02G037700.1) as orthologs found in blocks collinear with Spov3_chr4.04752.

[0415] Broad beans - Spov3_chr4.04752

[0416] Reference transcriptome = V.faba_csfl_reftrans v2 - available at pulsedb.org

[0417] Currently, there is no available genome for broad beans; instead, there is a transcriptome with assembled RNASeq data. Therefore, it is suitable for BLAST analysis but incompatible with Synteny Analyser.

[0418]

[0419] In the broad bean transcriptome, Spov3_chr4.04752 returned a single hit.

[0420] Table 8

[0421]

[0422] Example 9: Downy mildew resistance in sunflower plants

[0423] It is hypothesized that sunflowers possessing a functionally altered susceptibility gene (orthologous to SEQ ID NO: 4, 96, 172) or protein (orthologous to SEQ ID NO: 6, 98, or 174) will be less susceptible to downy mildew infection. To test this hypothesis, sunflower mutants with altered S gene expression (and protein translation) were generated through gene editing or chemical mutagenesis. These allele-homozygous mutants were then tested in a bioassay to determine the level of their reduced susceptibility to downy mildew infection. The results were compared to mutants with mutations in only one allele or plants with unmutated alleles. Plants were directly inoculated with more than one pathogenic axis downy mildew race (strain or isolate). Alternatively, seeds were sown in potting soil mixed with infected soil (soil known to cause disease in susceptible plants), or seedlings were transferred to such soil. Susceptibility levels were assessed in multiple ways. For example, infection levels can be determined by scoring the number of germinating seedlings or the percentage of diseased seedlings after sowing, establishing a disease grading score, or by confirming the presence of a pathogen through diagnostic tests. In all these tests, sunflower plants homozygous for the mutant allele are compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It has been demonstrated that sunflower plants homozygous for the mutant allele are less susceptible to downy mildew compared to variants carrying the unmutated (wild-type) allele.

[0424] Example 10: Downy mildew resistance in maize plants

[0425] It was hypothesized that maize plants possessing a functionally altered susceptibility gene (orthologous to SEQ ID NO: 4, 96, 172) or protein (orthologous to SEQ ID NO: 6, 98, or 174) would be less susceptible to downy mildew infection. To test this hypothesis, maize mutant plants with altered S gene expression (and protein translation) were generated through gene editing or chemical mutagenesis. These allele-homozygous mutants were then tested in a bioassay to determine the level of their reduced susceptibility to downy mildew infection. Results were compared with mutant plants mutated in only one allele or with unmutated alleles. More specifically, bioassays were performed on maize seedlings, detached leaves, or leaf discs. Plants were directly inoculated with races (strains or isolates) of more than one pathogen species of the genus *S.* (e.g., *Sorghi*, *Maydis*, *Sacchar*). Susceptibility levels were assessed in multiple ways. For example, infection levels can be determined by assessing the number of germinating seedlings after sowing or by scoring the percentage of diseased seedlings, establishing a disease grading score, or by confirming the presence of the pathogen through diagnostic tests. In all these tests, homozygous mutant allele maize plants are compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It has been demonstrated that homozygous mutant allele maize plants are less susceptible to downy mildew compared to the unmutated (wild-type) variant.

[0426] Example 11: Downy mildew resistance in sugar beet plants

[0427] It is hypothesized that sugar beets, red beets, and Swiss chard (all beet species) possessing altered functional susceptibility genes (orthologous to SEQ ID NO: 4, 96, 172) or proteins (orthologous to SEQ ID NO: 6, 98, or 174) will be less susceptible to downy mildew infection. To test this hypothesis, beet mutants with altered S gene expression (and protein translation) were generated through gene editing or chemical mutagenesis. These allele-homozygous mutants were then tested in a bioassay to determine the level of their reduced susceptibility to downy mildew infection. The results were compared to mutants with mutations in only one allele or plants with unmutated alleles. More specifically, bioassays were performed on beet seedlings, detached leaves, or leaf discs. Plants were directly inoculated with more than one pathogen, *Persona chinensis* (strains or isolates), by friction inoculation or spraying with a suspension of inoculum containing sporangia / spores. Susceptibility levels were assessed in multiple ways. For example, infection levels can be determined by scoring the number of germinating seedlings or the percentage of diseased seedlings after sowing, establishing a disease grading score, or by confirming the presence of a pathogen through diagnostic tests. In all these tests, beet plants homozygous for the mutant allele are compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It has been demonstrated that beet plants homozygous for the mutant allele are less susceptible to downy mildew compared to variants carrying the unmutated (wild-type) allele.

[0428] Example 12: Downy mildew resistance in lettuce plants

[0429] It is hypothesized that lettuce with altered functional susceptibility genes (orthologous to SEQ ID NO: 4, 96, 172) or proteins (orthologous to SEQ ID NO: 6, 98, or 174) will be less susceptible to downy mildew infection. To test this hypothesis, lettuce mutants with altered S gene expression (and protein translation) were generated through gene editing or chemical mutagenesis. These allele-homozygous mutants were then tested in a bioassay to determine the level of their reduced susceptibility to downy mildew infection. The results were compared with mutants with mutations in only one allele or with unmutated alleles. More specifically, bioassays were performed on lettuce seedlings, detached leaves, or leaf discs. Plants were directly inoculated with more than one pathogen, *Palmeria fusca* (strains or isolates), by friction inoculation or spraying with a suspension of inoculum containing sporangia / spores. Susceptibility levels were assessed in multiple ways. For example, infection levels can be determined by scoring the number of germinating seedlings after sowing or by assessing the percentage of diseased seedlings, establishing a disease grading score, or by confirming the presence of a pathogen through diagnostic tests. In all these tests, lettuce plants homozygous for the mutant allele are compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It has been demonstrated that lettuce plants homozygous for the mutant allele are less susceptible to downy mildew compared to variants carrying the unmutated (wild-type) allele.

[0430] Example 13: Downy mildew resistance in Cucurbitaceae

[0431] It is hypothesized that cucurbitaceous plants (e.g., cucumber, melon, watermelon, squash, pumpkin, bottle gourd, and other crops) possessing altered functional susceptibility genes (orthologous to SEQ ID NO: 4, 96, 172) or proteins (orthologous to SEQ ID NO: 6, 98, or 174) will be less susceptible to downy mildew infection. To test this hypothesis, mutant plants with altered S gene expression (and protein translation) were generated through gene editing or chemical mutagenesis. These allele-homozygous mutants were then tested in a bioassay to determine the level of reduced susceptibility to downy mildew infection. The results were compared to mutant plants with mutations in only one allele or plants with unmutated alleles. More specifically, bioassays were performed on seedlings, detached leaves, or leaf discs. Plants were directly inoculated with more than one pathogen, *Pseudomonas columbarium* (strains or isolates), by friction inoculation or spraying with a suspension of inoculum containing sporangia / spores. Susceptibility levels were assessed in multiple ways. For example, infection levels can be determined by scoring the number of germinating seedlings or the percentage of diseased seedlings after sowing, establishing a disease grading score, or by confirming the presence of a pathogen through diagnostic tests. In all these tests, plants homozygous for the mutant allele are compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It has been demonstrated that plants homozygous for the mutant allele are less susceptible to downy mildew compared to variants carrying the unmutated (wild-type) allele.

[0432] Example 14: Downy mildew resistance in cucumber plants

[0433] It is hypothesized that cucumbers possessing altered functional susceptibility genes (orthologous to SEQ ID NO: 4, 96, 172) or proteins (orthologous to SEQ ID NO: 6, 98, or 174) will be less susceptible to downy mildew infection. To test this hypothesis, cucumber mutants with altered S gene expression (and protein translation) were generated through gene editing or chemical mutagenesis. These allele-homozygous mutants were then tested in a bioassay to determine the level of their reduced susceptibility to downy mildew infection. The results were compared with mutants containing only one mutated allele or with unmutated alleles. More specifically, bioassays were performed on cucumber seedlings, detached leaves, or leaf discs. Plants were directly inoculated with more than one pathogen, *Pseudomonas columbarium* (strains or isolates), by friction inoculation or spraying with a suspension of inoculum containing sporangia / spores. Susceptibility levels were assessed in multiple ways. For example, infection levels can be determined by scoring the number of germinating seedlings or the percentage of diseased seedlings after sowing, or by using diagnostic tests to identify the presence of pathogens. In all these tests, cucumber plants homozygous for the mutant allele are compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It has been demonstrated that cucumber plants homozygous for the mutant allele are less susceptible to downy mildew compared to variants carrying the unmutated (wild-type) allele.

[0434] Example 15: Downy mildew resistance in other plant species

[0435] It is hypothesized that other plants possessing functionally altered susceptibility genes (orthologous to SEQ ID NO: 4, 96, 172) or proteins (orthologous to SEQ ID NO: 6, 98, or 174) will be less susceptible to downy mildew infection. These plants include, but are not limited to, carrots, legumes, arugula, basil, soybeans, sorghum, impatiens, wheat, barley, rye, peas, broad beans, Brassica species (e.g., but not limited to, rapeseed, cabbage, turnip, and shepherd's purse), and Solanaceae plants (e.g., but not limited to, tomatoes, peppers, and eggplants). To test this hypothesis, mutant plants with altered S gene expression (and protein translation) are generated through gene editing or chemical mutagenesis. These allele-homozygous mutants are then tested in a bioassay to determine the level of their reduced susceptibility to downy mildew infection. The results are compared to mutant plants with mutations in only one allele or plants with unmutated alleles. More specifically, bioassays were performed on seedlings, detached leaves, or leaf discs. Plants were directly inoculated with more than one pathogen race (strain or isolate) by friction inoculation or spraying with a suspension of inoculum containing sporangia / spores. The following crop / downy mildew combinations were tested: carrot / parasitic downy mildew; arugula / Peronospora erucastri; basil / Peronospora belbahrii; soybean / Northeast downy mildew; sorghum / Sorghum finger downy mildew; impatiens / Plasmopara obducen; wheat / Phytophthora macrosporum; barley / Phytophthora macrosporum; rye / Phytophthora macrosporum; pea / Phytophthora phaseoli; broad bean / Phytophthora phaseoli; Brassica species / Hyaloparonospora brassicae; and Solanaceae / Tobacco downy mildew. Susceptibility levels were assessed in multiple ways. For example, infection levels can be determined by scoring the number of germinating seedlings or the percentage of diseased seedlings after sowing, establishing a disease grading score, or by confirming the presence of a pathogen through diagnostic tests. In all these tests, plants homozygous for the mutant allele are compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It has been demonstrated that plants homozygous for the mutant allele are less susceptible to downy mildew compared to variants carrying the unmutated (wild-type) allele.

[0436] Example 16: Resistance to Phytophthora blight, which causes root, stem base, and fruit rot

[0437] Reduced susceptibility to other oomycete pathogens (most notably Phytophthora species) was determined in a similar manner. Plants homozygous for the mutant allele were compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It was demonstrated that plants homozygous for the mutant allele were less susceptible to Phytophthora compared to variants carrying the unmutated (wild-type) allele. The following crop / pathogen combinations were tested: tomato / Phytophthora pathogenicum (late blight); tomato / Phytophthora capsicum; capsicum / Phytophthora capsicum; eggplant / Phytophthora capsicum; tobacco / Phytophthora capsicum (Phytophthoranicotianae); and cucurbit crops (e.g., cucumber, squash, pumpkin, watermelon, and melon) / Phytophthora capsicum.

[0438] Example 17: Resistance to Pythium, a disease that causes damping-off and root rot

[0439] Reduced susceptibility to other oomycete pathogens, most notably *Pythium* species, was determined in a similar manner. Plants homozygous for the mutant allele were compared to plants with only one mutant allele (heterozygous) or plants carrying only the unmutated allele (homozygous wild-type). It was demonstrated that plants homozygous for the mutant allele were less susceptible to downy mildew compared to variants carrying the unmutated (wild-type) variant. The following crop / pathogen combinations were tested: carrot / *Pythium violae*, *Pythium virgaurea*, and *Pythium maximum*; Swiss chard / *Pythium violae*, *Pythium virgaurea*, and *Pythium maximum*; and spinach / *Pythium violae*, *Pythium virgaurea*, and *Pythium maximum*.

[0440] Preservation Information

[0441] A total of 625 seeds of the resistant M3 spinach strain “Nr22-18” were deposited on May 14, 2021, by KWSVegetables BV under accession number NCIMB 43773 at the NCIMB Ltd. Depository Centre (UK Food Industry and Marine Bacteria Culture Collection Centre), located at Ferguson House, Croft, AB21 9YA Bucksbum, Aberdeen, UK. During the pending period of this application, the depositary material will be available to the authorized person upon request or as determined by a professional solution pursuant to Rule 32EPC. Pursuant to 37 CFR §1.808(b), all restrictions on the public accessibility of the depositary material will be irrevocably lifted upon patent grant. The deposit will remain in place for 30 years, or 5 years after the most recent request, or for the period of enforceability of the patent, whichever is longer, and will be replaced if it becomes unusable within that period. The applicant does not waive any rights granted by this patent application or the Plant Variety Protection Act (7 USC 2321, etc.).

Claims

1. A method to reduce spinach ( Spinacia olerace a) A method for determining plant susceptibility to pathogens of spreading downy mildew or anthracnose, said method comprising: By targeting DNA modification or gene introgression to enhance the activity or expression of the susceptibility gene (S gene) encoding RPS2-like resistance protein, The S gene encodes an RPS2-like resistance protein with an amino acid sequence encoded by SEQ ID NO:

339.

2. The method as described in claim 1, wherein, The DNA modification produces a sequence selected from SEQ ID NO: 169 or 170.

3. The method as described in claim 1 or 2, wherein, The modification includes, and is flanked by, SEQ ID NO: 241 and SEQ ID NO:

251.

4. The method as described in claim 1 or 2, wherein, The DNA modification produces a sequence identical to that of SEQ ID NO:

342.

5. The method as described in claim 1 or 2, wherein, The anthrax pathogen belongs to the genus *Colletotrichum* (…). Colletotrichum ).

6. The method of claim 5, wherein, The pathogen is *Colletotrichum gloeosporioides*, a species of spinach. Colletotrichum dematium f. sp. Spinaciae ).

7. A spinach (Spinaciaoleracea) plant with reduced susceptibility to pathogens of the species *Peronospora* or *Colletotrichum*, said plant containing one or more nucleotide modifications at its gene locus, wherein, When compared with plants without the modification, the locus contains a susceptibility gene (S gene) encoding an RPS2-like resistance protein. The S gene encodes an RPS2-like resistance protein with an amino acid sequence encoded by SEQ ID NO:

339.

8. The plant as described in claim 7, wherein, The pathogen of the genus *Colletotrichum* is *Colletotrichum gloeosporioides*, a species of spinach.

9. The plant as described in claim 7, wherein, The one or more nucleotide modifications result in increased activity or expression of the protein encoded by the S gene. Optionally, the one or more nucleotide modifications include insertions, deletions, or single nucleotide polymorphisms (SNPs).

10. The plant as described in claim 7 or 9, wherein, The one or more nucleotide modifications produce a sequence selected from SEQ ID NO: 169 or 170.

11. The plant as described in claim 7 or 9, wherein, The locus contains and is flanked by SEQ ID NO: 241 and SEQ ID NO:

251.

12. The plant as described in claim 7 or 9, wherein, The modification produces a sequence identical to the sequence in SEQ ID NO:

342.

13. Plant cells derived from any one of claims 7 to 11.

14. A plant part derived from any one of claims 7 to 11.

Images