Plant resistance genes and means for their identification

Abstract

More effective breeding against Cercospora leaf spot or the development of new resistant lines is possible via the provision of Cercospora resistance mediated genes according to the present application; in particular, the dominant resistance effect in the target plant is caused by the properties of the single identified genes. The Cercospora resistance mediated genes as well as the aforementioned embodiments of the present application provide additional applications, for example the use of alleles of the resistance genes in cis- or transgenetic approaches with the aim to develop new resistant cultivars.

Description

Technical Field

[0001] This invention relates to nucleic acid molecules encoding polypeptides that confer resistance to the fungus *Cercosporabeticola* on plants, particularly sugar beet species expressing the polypeptide, and polypeptides encoded by nucleic acid molecules according to the invention. Specifically, the nucleic acid molecules according to the invention are characterized in that the resistance effect against *Cercospora* conferred by the polypeptide is dominant. Furthermore, the invention relates to plants resistant to *Cercospora*, plant cells, plant organs, plant tissues, plant parts, or seeds or offspring of plants containing the nucleic acid molecule or a portion thereof as an endogenous gene, as an editing gene, or as a transgenic gene. Furthermore, the invention also covers methods for increasing resistance to *Cercospora* in sugar beet species, and methods for producing or identifying, and possibly selecting, plants resistant to *Cercospora*. The invention also covers methods for monitoring infection by the pathogen *Cercospora*, and oligonucleotide probes and primers for hybridization with nucleic acid molecules according to the invention. Background Technology

[0002] Cercospora leaf spot is one of the most important globally prevalent foliage diseases affecting various plants, including sugar beets and spinach (Spinacia oleracea). It is caused by the fungus Cercospora betaecarpa. Infected plants typically develop small, relatively circular leaf spots (2-3 mm) with a light gray center surrounded by a reddish-brown edge. In severe infections, the spots overlap, causing the entire leaf to dry out. Smaller black spots (pseudostae) are visible within the fully formed spots, and under moist conditions, a gray felt-like covering (conidial carriers) forms, primarily on the lower part of the leaf. Severely infected leaves first turn yellow, then brown, and die. However, new leaves grow in parallel, during which the leaves become infected again and die. Initially, symptoms are only visible on individual plants; however, as the disease spreads, persistent nests of infection often form. Further spread occurs throughout the field via rain and wind. In the case of Swiss chard plants, damaged leaves are unacceptable to consumers, and even low disease stress can lead to severe crop shortages.

[0003] The pathogen *Cercospora beticola* was first described in Italy in the second half of the 19th century. Up to 40% of crop loss can occur due to severe infection, which may be caused by wet weather, early row closure, high infection potential in previous years, or heavy irrigation. These losses are caused by reduced sugar content and reduced sugar beet yield; see Holtschulte ((2000) "Cercospora beticola - worldwide distribution and incidence," pp. 5-16, in "Cercospora beticola Sacc. Biology, Agronomic Influence and Control Measures in Sugar Beet," Vol. 2 (edited by MJ Cassher, B. Holtschulte, MRMolard, F. Rosso, G. Steinrücken, R. Beckers). International Institute for Beet Research, Brussels, Belgium, p. 215). Intercropping or fungicides are frequently used to combat this disease. Chemical control of Cercospora beticola via fungicides is costly for farmers and pollutes the environment. Furthermore, chemical treatment of edible Swiss chard leaves reduces consumer acceptance. Repeated application of fungicides adds selective pressure on fungicide-resistant Cercospora beticola strains. This runs counter to sustainable agricultural practices. It is worth mentioning that in recent years, stems of Cercospora beticola resistant to one or more fungicides have emerged; see Trkulja, Nenad R. et al., "Molecular and experimental evidence of multi-resistance of Cercospora beticola field populations to MBC, DMI and QoI fungicides." European Journal of Plant Pathology 149.4 (2017):895-910. This problem has become so serious that the German Federal Office of Consumer Protection and Food Safety (BVL) has granted an exemption for copper-based fungicides used to control Cercospora. However, copper-based fungicides are generally considered (depending on dosage) to be harmful to humans and the environment. Copper is a heavy metal that can accumulate in soil.

[0004] Indirect resistance can be achieved by selecting cultivars with healthy leaves and cultivating sugar beets in a crop rotation of at least three years. Significantly better control of infection can be achieved using combinations of tolerant or resistant cultivars. Since 2000, commercially available sugar beet cultivars resistant to Cercospora (Steinrücken 1997, “Die Züchtung von Cercospora-resistenten Zuckerrüben.” [“The breeding of Cercospora-resistant sugar beets.”]) für Pflanzenzüchtung [Lectures on Plant Breeding], Vol. 37, Lecture symposium, March 4-5, 1997, Kiel). These cultivars exhibit quantitative resistance to *Cercosporabeticola*. The resistance in these cultivars is based on several genes and is quantitatively inherited, in which the exact number of genes responsible for resistance is unknown; see Weiland and Koch (2004), Sugarbeet leaf spot disease (Cercosporabeticola Sacc.), The Plant Journal, 5(3), 157-166. Complex quantitative inheritance was confirmed by analysis of several quantitative trait loci (QTLs). This method allows for mapping of polygenic inherited resistance and is a reliable technique for identifying the number and location of genetic resistance factors on the genetic linkage map of host plants. In this way, multiple pathogenic QTLs can be identified on each chromosome of sugar beets.

[0005] Plotting was performed using different Cercospora resistance donors, most of which showed small QTL effects. The claimed maximum phenotypic value was 5%.

[0006] In a series of studies, lists of differentially expressed genes were described. In the study by Weltmeier et al. ((2011) Transcript profiles in sugar beet genotypes uncover timing and strength of defense reactions to Cercospora beticola infection, Molecular plant-microbeinteractions, 24(7), 758-772), whole-genome expression profiles for various sugar beet genotypes (i.e., Cercospora resistance, tolerance, susceptibility, etc.) were created using microarray-based techniques during pathogen infection to analyze transcriptional changes related to leaf spot in the expression profiles. Through these analyses, the authors were able to create pathogen-induced transcriptional profiles and identify potential candidate genes in various test genotypes of sugar beet. However, these genes have not yet been characterized in detail. To date, the genetic and functional background of Cercospora resistance and the characteristics of resistance genes remain completely unclear.

[0007] However, through quantitative inheritance of QTLs, not only is desired resistance to *Cercospora beetroot* introduced into plants, but undesirable traits, such as reduced yield, are also often introduced due to the inheritance of additional genes associated with positive traits of *Cercospora* resistance. This phenomenon is also known as “linkage cascading.” Furthermore, the enormous breeding costs required to select multiple resistance loci without consequently reducing yield can negatively impact plant vigor; see Weiland and Koch, 2004.

[0008] For over a decade, breeding companies have offered cultivated varieties tolerant to *Cercospora*. The resistance in these cultivars is inherited through multiple resistance genes with minimal effects. However, a drawback of these cultivars is that their development is extremely laborious and complex due to genetic complexity, and they exhibit significantly poorer yield performance compared to normal cultivars in the absence of infection. This may be related, among other things, to the epigenetic interactions between some resistance genes and genes responsible for sugar production, leading to reduced plant adaptability in the absence of pathogens. Furthermore, *Cercospora* exhibits a tendency to overcome the tolerance of long-established cultivars. Moreover, resistance scores from unadapted wild-type genetic resources available to date are generally unreliable and incomparable to each other because potential studies have been conducted under different environmental conditions, different infection pressures, and on different pathogenic stems of *Cercospora*. In this regard, it should be mentioned that environmental parameters such as moisture, temperature, and wind (which tend to be unstable) have a significant impact on the progression of *Cercospora* diseases after infection. It is common for a particular genetic resource to show a high level of tolerance / resistance in one study, while tending to be completely susceptible in another. Due to the factors given above, a dominant resistance gene with a major effect against Cercospora has not yet been identified, although there is a strong need for such a gene—which could be easily transferred into existing cultivars and varieties to establish resistance to Cercospora.

[0009] Due to the complex genetics and the large number of genes involved in the development of resistance (most of which have not yet been identified and characterized), the use of new breeding techniques based on gene editing (e.g., with the aid of TALE nucleases or CRISPR systems, as well as transgenic methods) is not applicable to the genetic material available to date.

[0010] To achieve sustainable breeding against Cercospora leaf spot (i.e., eliminating the danger of overcoming resistance in Cercospora variants), it is necessary to continuously identify new resistance genes and integrate them into the gene pool of cultivated plants (such as sugar beets). In particular, the aim is to provide suitable resistance genes that, once expressed in plants, already exhibit a strong dominant resistance effect against *Cercospora beetans*. According to the invention, this objective is achieved through the embodiments characterized in the claims and specification.

[0011] Invention Summary

[0012] This invention relates to nucleic acid molecules capable of conferring resistance in plants (particularly red beets) to the genus *Cercospora* (particularly the fungus *Cercospora betaine*). This results in the production of polypeptides encoded by the nucleic acid molecules in the plant. The nucleic acid molecules that produce the polypeptides upon expression themselves already exhibit a strong dominant resistance effect against *Cercospora betaine* in the plant.

[0013] Furthermore, the present invention relates to plants resistant to the genus *Cercospora*, plant cells, plant organs, plant tissues, plant parts, seeds, seed stock, or plant progeny that endogenously or transgenic contain nucleic acid molecules or portions thereof. Depending on the specific optional embodiment, those plants and their components obtained via basic biological processes are excluded.

[0014] This invention also covers methods for increasing the plant resistance of sugar beet species to *Cercospora*, and methods for producing or identifying, and possibly selecting, plants resistant to *Cercospora*. This invention further covers methods for monitoring infection by the pathogen *Cercospora beetani*, and oligonucleotides as probes and primers for hybridization with nucleic acid molecules according to the invention.

[0015] Therefore, the present invention relates to embodiments listed in the following points and shown in the examples and drawings.

[0016] [1] A nucleic acid molecule encoding a polypeptide that confers resistance to the genus *Cercospora* to plants expressing the polypeptide, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from the following:

[0017] (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 3;

[0018] (b) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 2;

[0019] (c) A nucleotide sequence comprising a DNA sequence selected from SEQ ID No. 1 or SEQ ID No. 53;

[0020] (d) A nucleotide sequence that hybridizes under strict conditions with a nucleotide sequence complementary to the nucleotide sequence according to (a), (b), or (c);

[0021] (e) A nucleotide sequence that encodes a polypeptide that is different from the polypeptide encoded by the nucleotide sequence according to (a), (b) or (c) by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence;

[0022] (f) A nucleotide sequence encoding a polypeptide having at least 70% of the same amino acid sequence as the amino acid sequence according to SEQ ID No. 3;

[0023] (g) A nucleotide sequence that is at least 70% identical to the DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2;

[0024] The resistance to the genus Cercospora is preferably resistance to Cercospora betaecarpa, or the plant is preferably a subspecies of red beet (Beta vulgaris subsp. vulgaris), and particularly preferably a sugar beet.

[0025] [2] According to the nucleic acid molecule described in [1], the resistance effect against Cercospora conferred by the polypeptide is dominant in the plant, preferably wherein the polypeptide conferred at least one grade score, and preferably more than one grade score, particularly preferably at least two grade scores, particularly preferably at least three grade scores, and especially preferably at least four grade scores of resistance effect.

[0026] [3] The nucleic acid molecule according to [1] or [2] is characterized in that the nucleic acid molecule is derived from sea beet (Betavulgaris subsp. maritima).

[0027] [4] A polypeptide encoded by a nucleic acid molecule according to any one of [1] to [3].

[0028] [5] A vector or expression cassette comprising a nucleic acid molecule according to any one of [1] to [3], wherein the nucleic acid molecule is preferably heterologous to the vector or expression cassette.

[0029] [6] A cell comprising a nucleic acid molecule according to any one of [1] to [3], or a vector or expression cassette according to [5], wherein the nucleic acid molecule or expression cassette is preferably present as an endogenous genotype or transgene.

[0030] [7] A plant or part thereof resistant to *Ceratophyllum*, characterized in that the plant or part thereof contains a nucleic acid molecule or nucleotide sequence according to any one of [1] to [3], or a vector or expression cassette according to [5], wherein the plant endogenously containing the nucleic acid molecule is a sugar beet species (not *Begonia spp.*) or a red beet. The nucleic acid molecule or nucleotide sequence may be endogenously or transgenic. Seeds of the plant according to this section can be obtained from the NCIMB deposit in Aberdeen, UK, accession number NCIMB 43646. The plant according to this section can be obtained from the deposited seeds.

[0031] [8] The plant according to [7] is characterized in that the plant is a hybrid plant.

[0032] [9] The plant according to [7] or [8] is characterized in that the nucleic acid molecule exists in the plant genome in a heterozygous or homozygous form.

[0033]

[10] Seeds or progeny of a plant according to any one of [7] to [9], wherein the seed or progeny contains a nucleic acid molecule or nucleotide sequence according to any one of [1] to [3], or a vector or expression cassette according to [5]. The nucleic acid molecule may be transgenic, non-transgenic, or endogenously present. Seeds according to this paragraph may be obtained from the NCIMB deposit in Aberdeen, UK, accession number NCIMB 43646.

[0034]

[11] A method for increasing plant resistance to Cercospora, comprising the following steps:

[0035] (i) Integrating a nucleic acid molecule according to one of [1] to [3], or a vector or expression cassette according to [5], into the genome of at least one cell of a plant by means of homologous directed repair or homologous recombination (preferably supported by site-directed nucleases), and optionally regenerating a plant from at least one plant cell; or

[0036] (ii) Particularly after infection with *Cercospora*, preferably by modifying, for example, a natural promoter containing the DNA sequence according to SEQ ID No. 7, or by linking a nucleic acid molecule according to any one of [1] to [3] to a heterologous promoter having a higher activity level than, for example, a natural promoter containing the DNA sequence according to SEQ ID No. 7, thereby increasing the expression of the nucleic acid molecule according to any one of [1] to [3] in at least one cell of the plant, and optionally regenerating the plant from at least one plant cell; or

[0037] (iii) In at least one cell of a plant, the activity and / or stability of the polypeptide according to [4] is increased by modifying the nucleotide sequence of a nucleic acid molecule according to any one of [1] to [3], and optionally the plant is regenerated from at least one plant cell; or

[0038] (iv) Transforming plant cells with a nucleic acid molecule according to one of [1] to [3], or a vector or expression cassette according to [5], and optionally regenerating (transgenic) plants from the transformed plant cells;

[0039] The resistance to Cercospora is preferably resistance to Cercospora beetroot, or the plant is preferably a sugar beet species, preferably red beet, and especially sugar beet.

[0040]

[12] A method for producing resistant Cercospora plants according to any one of [7] to [9], the method comprising the following steps:

[0041] (a) Transforming plant cells with a nucleic acid molecule according to any one of [1] to [3], or a vector or expression cassette according to [5]; and

[0042] (b) Regeneration of transgenic plants from transformed plant cells; or

[0043] (i) Introducing a site-directed nuclease and a repair matrix into cells of sugar-grown sugar beet species, wherein the site-directed nuclease is capable of generating at least one double-strand break of DNA in the cell genome (preferably upstream and / or downstream of the target region), and the repair matrix comprises a nucleic acid molecule according to one of [1] to [3];

[0044] (ii) culturing cells derived from (i) under conditions allowing homologous directed repair or homologous recombination, wherein nucleic acid molecules are introduced from the repair matrix into the plant genome; and

[0045] (iii) Cell regeneration plants modified in (ii).

[0046]

[13] The method according to

[12] is characterized in that the target region comprises an allelic variant of a nucleic acid molecule according to one of [1] to [3], wherein the allelic variant encodes a polypeptide that does not confer resistance or mild resistance to Cercospora.

[0047]

[14] The method according to

[12] or

[13] is characterized in that the at least one double-strand break occurs at a position up to 10,000 base pairs upstream and / or downstream of the target region, or at a position up to 10,000 base pairs away from the allele variant as defined in

[13] .

[0048]

[15] The method according to

[12] or

[13] is characterized in that the allelic variant of the nucleic acid molecule comprises a nucleotide sequence selected from:

[0049] (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 6;

[0050] (b) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 5;

[0051] (c) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 4;

[0052] (d) A nucleotide sequence that hybridizes under strict conditions with a nucleotide sequence complementary to the nucleotide sequence according to (a), (b), or (c);

[0053] (e) A nucleotide sequence encoding a polypeptide that differs from a polypeptide encoded by a nucleotide sequence according to (a), (b), or (c) via substitutions, deletions, and / or additions of one or more amino acids in its amino acid sequence; or

[0054] (f) A nucleotide sequence encoding a polypeptide having at least 80% of the same amino acid sequence as the amino acid sequence according to SEQ ID No. 6.

[0055]

[16] A plant or a part thereof, which is obtained or may be obtained according to any one of the methods described in

[12] to

[15] .

[0056]

[17] A method for identifying and optionally providing sugar beet species resistant to Cercospora, characterized in that the method comprises at least step (i) or (ii):

[0057] (i) Detecting in a plant or a portion of a plant the presence and / or expression of a nucleic acid molecule according to one of [1] to [3], or the presence of a polypeptide according to [4]; and / or

[0058] (ii) Detecting at least one marker locus in the nucleotide sequence or co-segregating region of a nucleic acid molecule according to any one of [1] to [3]; and

[0059] (iii) Possible selection of plants resistant to Cercospora beetroot

[0060] The detection in step (i) or step (ii) is based on the use of at least one molecular marker.

[0061]

[18] A method for identifying nucleic acid molecules that encode polypeptides capable of conferring resistance to Cercospora spp. on sugar beet species expressing polypeptides, characterized in that the method comprises the following steps:

[0062] (i) Compare the amino acid sequence of the polypeptide according to [4] with an amino acid sequence from a sequence database, or identify allelic variants encoding the polypeptide according to [4] in the genotype of a sugar beet species;

[0063] (ii) Identify an amino acid sequence or an allelic variant encoding an amino acid sequence, wherein the amino acid sequence is at least 80% identical to the amino acid sequence of the polypeptide according to [4];

[0064] (iii) Introducing a nucleic acid molecule or allelic variant encoding the identified amino acid sequence into a sugar beet species and expressing the nucleic acid molecule in the plant; and

[0065] (iv) Detect resistance to Cercospora.

[0066]

[19] A method for cultivating sugar beet species, the method comprising:

[0067] (i) Providing plants according to any one of [7] to [9], cultivating granulated seeds of sugar beet plants or genus Beta plants according to any one of

[26] to

[39] , producing plants of sugar-producing cultivated sugar beet species by means of any one of

[12] to

[15] , or identifying and selecting genus Beta plants by means of the method according to

[17] , and

[0068] (ii) To cultivate plants or their offspring derived from (i),

[0069] This method helps prevent infection of cultivated plants by the genus Cercospora.

[0070]

[20] An oligonucleotide of at least 15, 16, 17, 18, 19 or 20 nucleotides in length, preferably at least 21, 22, 23, 24 or 25, particularly preferably at least 30, 35, 40, 45 or 50, and especially preferably at least 100, 200, 300 or 500 nucleotides, which hybridizes with a nucleotide sequence as defined in one of [1] to [3].

[0071]

[21] A pair of oligonucleotides, preferably oligonucleotides according to

[20] or kits containing such oligonucleotides, wherein the oligonucleotides are suitable as forward and reverse primers for hybridization with regions in the sugar beet genome that are conferred by the polypeptide according to [4] or co-segregated with nucleic acid molecules according to one of [1] to [3].

[0072]

[22] Use of the nucleic acid molecules described in any one of [1] to [3] in producing plants resistant to the genus *Cercospora* (red beet).

[0073]

[23] A method for producing an organism comprising a mutant form according to [1] and / or a promoter comprising a nucleic acid sequence selected from:

[0074] (a)SEQ ID NO:7

[0075] (b) A nucleotide sequence that hybridizes under stringent conditions to a sequence complementary to the sequence in (a).

[0076] (c) A nucleotide sequence that is at least 70% identical to the sequence according to SEQ ID NO:7

[0077] The method includes the following steps:

[0078] (I) Providing an organism or cell containing nucleic acid molecules and / or promoters

[0079] (II) Increase the mutation rate of an organism or cell, or induce mutations in an organism or cell.

[0080] (III) Select an organism's phenotype that, as a result of mutation, exhibits altered resistance or altered resistance levels to *Cercospora beetroot*, or select an organism or cell genotype containing a mutation in a nucleic acid molecule and / or promoter, wherein the mutation is generated via step (II), and optionally...

[0081] (IV) Cell regeneration organism obtained via step (III).

[0082]

[24] The method according to

[23] , wherein the organism is a plant.

[0083]

[25] The method according to

[24] , wherein the plant is a sugar beet, preferably red beet, more preferably sugar beet or red beet.

[0084]

[26] A granulated seed of a sugar beet plant or a beet genus (including red beet) containing a nucleic acid molecule according to [1].

[0085]

[27] According to

[26] the granulated seeds, wherein the beet body is suitable as a raw material for industrial sugar production or for food consumption.

[0086]

[28] The granulated seeds according to

[26] or

[27] , wherein the granulated seeds are monoembryonic.

[0087]

[29] The granulated seeds according to

[26] to

[28] , wherein sugar beet plants can be harvested before bolting.

[0088]

[30] The granulated seeds according to

[26] to

[29] , wherein the resistance to Cercospora is resistance to Cercospora betaecarpa.

[0089]

[31] The granulated seeds according to

[26] to

[30] , wherein the sugar beet plant is biennial.

[0090]

[32] The granulated seeds according to

[26] to

[31] and

[124] have undergone technical treatment, wherein the technical treatment is selected from:

[0091] (a) Polishing;

[0092] (b) Seed dressing;

[0093] (c) crusting; and

[0094] (d) Coloring.

[0095]

[33] The granulated seeds according to

[26] to

[32] , wherein the pellets contain at least one chemical selected from:

[0096] (a) Insecticides;

[0097] (b) fungicides; and

[0098] (c) Fertilizing.

[0099]

[34] Granulated seeds according to

[26] to

[33] , wherein the seeds have undergone initiation or germination before or during granulation.

[0100]

[35] The granulated seeds according to

[26] to

[34] , wherein the sugar beet plant is a hybrid sugar beet plant.

[0101]

[36] The granulated seeds according to

[26] to

[35] , wherein the nucleotide sequence includes at least one mutation.

[0102]

[37] The granulated seeds according to

[36] , wherein at least one mutation is a mutation relative to SEQ ID No. 1 or SEQ ID No. 2.

[0103]

[38] The granulated seeds according to

[36] or

[37] , comprising at least one mutated nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 99% identical to the amino acid sequence according to SEQ ID No. 3.

[0104]

[39] The granulated seeds according to

[38] include at least one mutated nucleotide sequence encoding a polypeptide having an amino acid sequence according to SEQ ID No. 3.

[0105]

[40] A package containing granulated seeds according to

[26] to

[39] or containing a primary species comprising nucleic acid molecules according to [1], wherein the primary species is preferably a primary species of a beet plant.

[0106]

[41] A mixture of granulated clumps and sugar beet seeds, wherein the sugar beet seeds contain a nucleic acid sequence encoding a polypeptide capable of conferring resistance to Cercospora, wherein the nucleotide sequence is selected from:

[0107] (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 3;

[0108] (b) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 2;

[0109] (c) A nucleotide sequence comprising a DNA sequence selected from SEQ ID No. 1 or SEQ ID No. 53;

[0110] (d) A nucleotide sequence that hybridizes under strict conditions with a nucleotide sequence complementary to the nucleotide sequence according to (a), (b), or (c);

[0111] (e) A nucleotide sequence that encodes a polypeptide that is different from the polypeptide encoded by the nucleotide sequence according to (a), (b) or (c) by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence;

[0112] (f) A nucleotide sequence encoding a polypeptide having at least 70% of the same amino acid sequence as the amino acid sequence according to SEQ ID No. 3;

[0113] (g) A nucleotide sequence that is at least 70% identical to the DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2.

[0114]

[42] A method for producing granulated sugar beet seeds according to

[26] to

[39] , the method comprising the following steps:

[0115] a) Provide sugar beet seeds containing a nucleic acid sequence encoding a polypeptide that confers resistance to Cercospora.

[0116] The nucleotide sequence is selected from:

[0117] (i) a nucleotide sequence encoding a polypeptide having at least 95% identical amino acid sequence to that of the amino acid sequence according to SEQ ID No. 3; and

[0118] (ii) A nucleotide sequence that is at least 95% identical to the DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2.

[0119] b) The sugar is encapsulated in granular aggregates using beet seeds.

[0120] c) Allow granular agglomerates to dry or allow granular agglomerates to dry.

[0121]

[43] The granulated seeds according to

[36] to

[39] contain at least one mutated nucleotide sequence that is an artificial nucleotide sequence that is not naturally occurring.

[0122]

[44] According to the method described in

[23] , mutagenic chemicals such as EMS or mutagenic radiation are applied during step (II).

[0123]

[45] A variety or cultivar of the genus *Betula* containing the nucleic acid molecule described in [1], or granulated seeds of such variety or cultivar.

[0124]

[46] A leaf of a Swiss chard plant containing nucleic acid molecules as described in [1].

[0125]

[47] A bag, preferably a plastic bag, comprising one or more leaves of the chard plant as described in

[46] .

[0126]

[48] ​​A method for identifying plants resistant to or tolerant of Cercospora, preferably sugar beet species or red beet subspecies, characterized in that the method comprises the following steps:

[0127] (i) Detecting in a plant or a portion of a plant the presence and / or expression of a nucleic acid molecule as described in [1] or a nucleotide sequence as defined in [1], or the presence of a polypeptide encoded by a nucleic acid molecule as described in [1] or a nucleotide sequence as defined in [1]; and / or

[0128] (ii) Detect at least one marker locus in the nucleotide sequence or co-separation region of the nucleic acid molecule as described in [1].

[0129] The co-segregating region is a genomic region co-segregating with Cercospora resistance conferred by a polypeptide, or with a nucleic acid molecule or nucleotide sequence, and wherein the co-segregating region comprises and preferably flanked by markers s4p1395s01 and s4p0421s01. More preferably, the co-segregating region comprises and flanked by marker pairs selected from: sxi0123s02 and s4p0238s01, s4p1396s01 and sxh1195s02, s4p1398s01 and s4p0234s01, s4p1462s01 and s4p0232s01, s4p1464s01 and s4p0323s01, s4p0044s01 and s4p0322s01, s4p0056s01 and s4p0320s01, s4p0058s01. 1 and sxh0942s04, s4p0059s01 and sxh1834s05, s4p1564s01 and s4p0317s01, s4p1998s01 and s4p4308s01, s4p1343s01 and s4p4305d01, s4p1408s01 and sxh0678s01, s4p1565s01 and s4p4301s01, s4p1409s01 and s4p8772s01, s4p1411s01 and s4p4295s01. Most preferably, the co-separation region comprises and has flanks selected from the following marker pairs: s4p1343s01 and s4p0421s01, s4p1408s01 and s4p0238s01, s4p1565s01 and sxh1195s02 s4p1409s01 and s4p0234s01, s4p1411s01 and s4p0232s01, s4p1414s01 and s4p0323s01, s4p1485s01 and s4p0322s01, s4p0257s01 and s4p0320s01, s4p0258s01 and sxh0942s04, s4p0260s01 and sxh1834s05, sxh0876s05 and s4p0317s01, s4p0262s01 and s4p4308s01, s4p0263s01 and s4p4305d01. For drawing purposes, the following label pairs are recommended: s4p4293s01 and s4p8772s01, preferably s4p4295s01 and s4p8772s01. The structural characteristics of the labels are given in Table 1B. The method may involve one or more of the following steps:

[0130] - Provide at least one plant, its tissue, seed, or at least one cell thereof.

[0131] - Extract DNA, preferably genomic DNA, from at least one plant, its tissue, seed, or at least one cell thereof.

[0132] - Perform detection on the extracted DNA

[0133] - Select for plants resistant to the genus Cercospora.

[0134]

[49] The method according to any one of

[48] or

[104] -

[113] is characterized in that the method comprises the following additional step:

[0135] - Create electronically transferable and / or electronically storeable data representing the detection of the presence of nucleic acid molecules or nucleotide sequences.

[0136]

[50] The method according to

[49] is characterized in that the method comprises the following additional step:

[0137] - Store data on a computer-readable medium.

[0138]

[51] The method according to one of

[48] to

[50] , wherein the marker is a molecular marker and / or a diagnostic marker.

[0139]

[52] A procedure for identifying plants exhibiting resistance or tolerance to the genus *Cercospora*, the procedure comprising detecting at least one polymorphism in the plant by at least one marker, preferably a single nucleotide polymorphism, wherein at least one marker is located in a chromosomal region flanked by two markers s4p1395s01 and s4p0421s01 or in a chromosomal region flanked by a pair of markers disclosed in

[48] , and wherein the plant contains a nucleic acid molecule or nucleotide sequence according to [1]. The marker may be a diagnostic and / or molecular marker. The procedure may involve one or more of the following steps:

[0140] - Provide at least one plant, its tissue, seed, or at least one cell thereof.

[0141] - Extract DNA, preferably genomic DNA, from at least one plant, its tissue, seed, or at least one cell thereof.

[0142] - Perform tests on the extracted DNA, and

[0143] - Select for plants resistant to the genus Cercospora.

[0144]

[53] According to the procedure described in

[52] , wherein

[0145] (i) The at least one single nucleotide polymorphism is genetically linked to a nucleic acid molecule or nucleotide sequence according to [1], or has a recombination frequency of about 10%, preferably 5%, more preferably 1% or lower with a nucleic acid molecule or nucleotide sequence according to [1], or

[0146] (ii) The at least one single nucleotide polymorphism is located within 2562 kbp, 2300 kbp, 2100 kbp, 1900 kbp, 1700 kbp, 1500 kbp, 1300 kbp, 1100 kbp, 900 kbp, 700 kbp, 500 kbp, 300 kbp, 100 kbp, 50 kbp, 25 kbp, 10 kbp, 5 kbp or 1 kbp or less of the nucleic acid molecule or nucleotide sequence according to [1].

[0147]

[54] The procedure or method according to any one of

[48] ,

[52] ,

[104] -

[113] , wherein the plant exhibits increased resistance or tolerance to Cercospora compared to plants lacking the nucleic acid molecule or nucleotide sequence described in [1].

[0148]

[55] The procedure according to any one of

[52] -

[54] ,

[57] -

[70] ,

[106] -

[113] , wherein a single nucleotide polymorphism is a nucleotide whose presence is genetically and / or statistically linked to the presence of a nucleic acid molecule or nucleotide sequence according to [1].

[0149]

[56] According to the procedure described in

[52] -

[55] , at least one of the markers is a marker given in Table 1b, or at least one of the markers is a pair of markers as disclosed in

[48] .

[0150]

[57] A procedure for identifying plants resistant to or tolerant to the genus *Cercospora*, the procedure comprising detecting at least two single nucleotide polymorphisms in or in the DNA of a plant by at least two markers, wherein at least one of the markers is located above or within a first chromosomal region flanked by markers selected from and a nucleotide sequence according to [1]:

[0151] s4p1395s01, sxi0123s02, s4p1396s01, s4p1398s01, s4p1462s01, s4p1464s01, s4p0044s01, s4p00 56s01, s4p0058s01, s4p0059s01, s4p1564s01, s4p1998s01, s4p1343s01, s4p1408s01, s4p1565s01, s4p1409s01, s4p1411s01, s4p1414s01, s4p1485s01, s4p0257s01, s4p0258s01, s4p0260s01, sxh08 76s05, s4p0262s01, s4p0263s01, s4p0264s01, s4p2271s01, s4p4288s01, s4p4290d01, s4p4293s01,

[0152] Furthermore, at least one of the markers is located above or within a second chromosomal region, the second chromosomal region being flanked by the polynucleotide according to claim 1 and markers selected from:

[0153] s4p0421s01, s4p0238s01, sxh1195s02, s4p0234s01, s4p0232s01, s4p0323s01, s4p03 22s01, s4p0320s01, sxh0942s04, sxh1834s05, s4p0317s01, s4p4308s01, s4p4305d01.

[0154]

[58] The procedure according to

[57] , wherein the at least two markers and the nucleotide sequence according to [1] are flanked by the first and second chromosomal regions, rather than as part of the first or second chromosomal region.

[0155]

[59] The procedure according to

[57] or

[58] , wherein each of the at least two markers is located within 2562 kbp, 2300 kbp, 2100 kbp, 1900 kbp, 1700 kbp, 1500 kbp, 1300 kbp, 1100 kbp, 900 kbp, 700 kbp, 500 kbp, 300 kbp, 100 kbp, 50 kbp, 25 kbp, 10 kbp, 5 kbp or 1 kbp or less of the nucleic acid molecule or nucleotide sequence according to [1].

[0156]

[60] According to the procedure described in

[57] -

[59] , at least one marker is located on a genomic region having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID No. 74 or SEQ ID No. 75, wherein the identity is preferably over the full length of SEQ ID No. 74 or SEQ ID No. 75.

[0157]

[61] According to the procedure described in

[57] -

[60] , wherein one of the at least two markers is located on a genomic region having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID No. 74, and one of the at least two markers is located on a genomic region having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID No. 75, wherein the identity is preferably over the entire length of SEQ ID No. 74 or SEQ ID No. 75.

[0158]

[62] The procedure according to

[57] -

[61] , wherein at least one of the at least two markers is located within 2562 kbp, 2300 kbp, 2100 kbp, 1900 kbp, 1700 kbp, 1500 kbp, 1300 kbp, 1100 kbp, 900 kbp, 700 kbp, 500 kbp, 300 kbp, 100 kbp, 50 kbp, 25 kbp, 10 kbp, 5 kbp or 1 kbp or less of the nucleic acid molecule or nucleotide sequence according to [1].

[0159]

[63] The procedure according to

[57] -

[62] , wherein each of the at least two markers is located within 2562 kbp, 2300 kbp, 2100 kbp, 1900 kbp, 1700 kbp, 1500 kbp, 1300 kbp, 1100 kbp, 900 kbp, 700 kbp, 500 kbp, 300 kbp, 100 kbp, 50 kbp, 25 kbp, 10 kbp, 5 kbp or 1 kbp or less of the nucleic acid molecule or nucleotide sequence according to [1].

[0160]

[64] The procedure described in

[57] -

[63] involves the detection of at least two single nucleotide polymorphisms in a first chromosomal region and at least two single nucleotide polymorphisms in a second chromosomal region, wherein each single nucleotide polymorphism is detected by a different marker.

[0161]

[65] The procedure described in

[57] -

[64] includes the following steps:

[0162] - Provide at least one plant, its tissue, seed, or at least one cell thereof.

[0163] - Extract DNA, preferably genomic DNA, from at least one plant, its tissue, seed, or at least one cell thereof.

[0164] - Perform the detection steps defined in any of

[57] -

[64] on the extracted DNA.

[0165]

[66] The procedure or method described in

[48] -

[65] includes a detection step comprising polymerase chain reaction (PCR).

[0166]

[67] The procedure or method described in

[48] -

[65] or

[112] or

[113] involves a polymerase chain reaction (PCR), wherein the PCR involves two allele-specific forward primers (or their use), and wherein the detection step involves fluorescence resonance energy transfer (FRET), wherein the presence, absence, or type of fluorescence is determined by a sensor. The sensor signal can be converted into electronically transferable and / or electronically storable data representing the detection of the presence of a nucleic acid molecule or nucleotide sequence according to [1]. Furthermore, the electronically transferable and / or electronically storable data can be stored on a computer-readable medium. The type of fluorescence can be its color / wavelength or a specific dye responsible for the fluorescence (such as FAM or HEX).

[0167]

[68] The procedure described in

[67] also relates to a universal reverse primer, or to the use of a universal reverse primer. Examples of universal primers are given in Table 1b.

[0168]

[69] The procedure according to any one of

[67] ,

[68] ,

[112] and

[113] , wherein the measurement of fluorescence by the sensor is an endpoint fluorescence reading.

[0169]

[70] The procedure according to any one of

[67] or

[69] ,

[112] and

[113] , wherein each of the two allele-specific forward primers contains a unique tail sequence that undergoes chemical binding with a specific FRET cassette during PCR.

[0170]

[71] A method for identifying plants resistant to or tolerant of Cercospora, preferably sugar beet species or red beet subspecies, characterized in that the method comprises the following steps:

[0171] - Provide at least one plant, its tissue, seed, or at least one cell thereof.

[0172] - Extract DNA from at least one plant, its tissue, or at least one cell thereof, preferably genomic DNA, and

[0173] - The presence or absence of nucleic acid molecules or nucleotide sequences as described in [1] is detected, preferably by markers as given in Table 1b, or by marker pairs as disclosed in

[48] , by oligonucleotide pairs as described in

[20] ,

[21] ,

[72] or

[73] or

[76] , by a set of three oligonucleotides as described in

[74] or

[75] , or by a molecular marker or a set of molecular markers as described in

[84] .

[0174] -Optionally select plants whose extracted DNA contains nucleic acid molecules or nucleotide sequences as described in [1].

[0175]

[72] A pair of oligonucleotides suitable for use as primers in PCR, capable of hybridizing with a genomic spacer region containing and flanking the markers s4p1395s01 and s4p0421s01. Preferably, the genomic spacer region contains and flanks the marker pair selected from: sxi0123s02 and s4p0238s01, s4p1396s01 and sxh1195s02, s4p1398s01 and s4p0234s01, s4p1462s01 and s4p0232s01, s4p1464s01 and s4p0323s01, s4p0044s01 and s4p0322s01, s4p0056s01 and s4p0320s01, s4p0058s01. 1 and sxh0942s04, s4p0059s01 and sxh1834s05, s4p1564s01 and s4p0317s01, s4p1998s01 and s4p4308s01, s4p1343s01 and s4p4305d01, s4p1408s01 and sxh0678s01, s4p1565s01 and s4p4301s01, s4p1409s01 and s4p8772s01, s4p1411s01 and s4p4295s01. Most preferably, the genomic spacer region contains and is flanked by marker pairs selected from the following: s4p1343s01 and s4p0421s01, s4p1408s01 and s4p0238s01, s4p1565s01 and sxh1195s02, s4p1409s01 and s4p0234s01, s4p1411s01 and s4p0232s01, s4p1414s01 and s4p0323s 01, s4p1485s01 and s4p0322s01, s4p0257s01 and s4p0320s01, s4p0258s01 and sxh0942s04, s4p0260s01 and sxh1834s05, sxh0876s05 and s4p0317s01, s4p0262s01 and s4p4308s01, s4p0263s01 and s4p4305d01. Genomic spacers can be genomic spacers of sugar beet species or red beet subspecies.

[0176]

[73] A pair of oligonucleotides suitable for use as primers in PCR, which are capable of hybridizing with a nucleic acid sequence according to SEQ ID No. 74 or SEQ ID No. 75, or with a sequence that is at least 99% identical in full length to the sequence according to SEQ ID No. 74 or SEQ ID No. 75.

[0177]

[74] A set of three oligonucleotides suitable for use as primers in PCR, comprising two forward primers and one reverse primer, wherein each primer has a different nucleotide sequence and wherein only one of the reverse primers and the two forward primers is capable of hybridizing with a genomic spacer region containing and flanking the markers s4p1395s01 and s4p0421s01. Preferably, the genomic spacer region contains and is flanked by marker pairs selected from the following: sxi0123s02 and s4p0238s01, s4p1396s01 and sxh1195s02, s4p1398s01 and s4p0234s01, s4p1462s01 and s4p0232s01, s4p1464s01 and s4p0323s01, s4p0044s01 and s4p0322s01, s4p0056s01 and s4p0320s01, s4p0058s0 1 and sxh0942s04, s4p0059s01 and sxh1834s05, s4p1564s01 and s4p0317s01, s4p1998s01 and s4p4308s01, s4p1343s01 and s4p4305d01, s4p1408s01 and sxh0678s01, s4p1565s01 and s4p4301s01, s4p1409s01 and s4p8772s01, s4p1411s01 and s4p4295s01. Most preferably, the genomic spacer region contains and is flanked by marker pairs selected from the following: s4p1343s01 and s4p0421s01, s4p1408s01 and s4p0238s01, s4p1565s01 and sxh1195s02, s4p1409s01 and s4p0234s01, s4p1411s01 and s4p0232s01, s4p1414s01 and s4p0323s 01, s4p1485s01 and s4p0322s01, s4p0257s01 and s4p0320s01, s4p0258s01 and sxh0942s04, s4p0260s01 and sxh1834s05, sxh0876s05 and s4p0317s01, s4p0262s01 and s4p4308s01, s4p0263s01 and s4p4305d01.

[0178]

[75] A set of three oligonucleotides suitable for use as primers in PCR, comprising two forward primers and one reverse primer, wherein each primer has a different nucleotide sequence, and wherein the reverse primer and only one of the two forward primers are capable of hybridizing with the sequence according to SEQ ID No. 74 or SEQ ID No. 75, or a sequence that is at least 99% identical in full length to the sequence according to SEQ ID No. 74 or SEQ ID No. 75.

[0179]

[76] The oligonucleotide pairs or groups according to

[72] -

[75] , wherein each oligonucleotide comprises or consists of 15-40 nucleotides, preferably 17-30 nucleotides, more preferably 19-25 nucleotides.

[0180]

[77] A procedure for quantifying the level of resistance or tolerance of plants to the genus *Cercospora*, the procedure comprising the following steps:

[0181] - Provide at least one first plant or at least one seed of a first plant containing at least one nucleic acid molecule or nucleotide sequence according to [1] and at least one second plant or at least one seed of a second plant lacking at least one nucleic acid molecule or nucleotide sequence according to [1].

[0182] - Cultivate plants or allow seeds to germinate and cultivate germinating plants, wherein a) the cultivation conditions allow for interaction with the genus *Cercospora*, or b) the plants are inoculated with *Cercospora*.

[0183] - Determine the infected surface area of ​​at least one leaf of each of the two plants.

[0184] -Optionally compare the resistance or tolerance level of the first plant with the resistance or tolerance level of the second plant, and / or determine the grade rating of the first and / or second plants selected from Table 1A.

[0185]

[78] The procedure according to

[77] is wherein the cultivation step is replaced by inoculating the leaves of the first plant and the leaves of the second plant in a solution containing Cercospora or in a solution containing Cercospora.

[0186]

[79] A method for reducing the application of fungicidal agricultural chemicals, the method comprising the following steps:

[0187] I) Provide seeds or original species containing nucleic acid molecules or nucleotide sequences as described in [1],

[0188] II) Planting or sowing seeds or primary varieties to allow them to germinate.

[0189] III) Cultivate germinating plants derived from step II),

[0190] IV) Harvest the plants cultivated during step III), or harvest plant parts such as storage organs, taproots, or harvest the seeds of the plants cultivated during step III).

[0191] This reduces or avoids the application of fungicidal agricultural chemicals during step II) and / or step III).

[0192]

[80] According to the method of

[79] , the fungicidal agricultural chemicals are effective against Cercospora, and these agricultural chemicals may include those containing one or more of the following fungicides: flutriafol, azoxystrobin, thiophanate-methyl, mancozeb, and those fungicides effective against Cercospora mentioned elsewhere herein.

[0193]

[81] The method according to

[79] or

[80] wherein the application of fungicidal agrochemicals during steps II) and / or III) is reduced or avoided compared to the cultivation of plants lacking nucleic acid molecules according to [1] under the same or equivalent conditions as the plants in step III).

[0194]

[82] The use of markers or molecular markers for the identification and / or selection of plants containing nucleic acid molecules or nucleotide sequences as described in [1].

[0195]

[83] According to the use described in

[82] , wherein the marker or molecular marker is used in the procedure or method described in

[48] -

[71] .

[0196]

[84] A molecular marker or a set of molecular markers—preferably covering two or three molecular markers—is applicable to the procedures or methods described in

[48] -

[71] .

[0197]

[85] A method for expressing a nucleic acid molecule or nucleotide sequence according to [1], the method comprising the steps of:

[0198] i) Introducing nucleic acid molecules or nucleotide sequences according to [1] into one or more cells,

[0199] ii) Cultivate one or more cells under conditions that allow for the proliferation of one or more cells and / or allow for the expression of nucleic acid molecules or nucleotide sequences as described in [1].

[0200]

[86] The method according to

[85] , wherein the method is a method for in vitro expression.

[0201]

[87] The method according to

[85] or

[86] , wherein the introduction in step i) is by conversion, transfection, electroporation or permeation.

[0202]

[88] The method according to

[85] -

[87] , wherein the introduction of a nucleic acid molecule or nucleotide sequence is the introduction of a vector, plasmid or transfer DNA (tDNA).

[0203]

[89] The method according to

[85] -

[88] , wherein the nucleic acid molecule or nucleotide sequence is part of the expression cassette and is operatively linked to an active, partially active or inducible promoter in one or more cells of i). The expression cassette may be the expression cassette according to [5].

[0204]

[90] The method according to

[89] wherein the promoter has at least 95% sequence identity with the 35S promoter derived from cauliflower mosaic virus, or the 35S promoter derived from cauliflower mosaic virus.

[0205]

[91] The method according to

[90] wherein the 35S promoter derived from cauliflower mosaic virus comprises or consists of a nucleic acid sequence according to SEQ ID No. 225.

[0206]

[92] According to the method described in

[85] -

[91] , one or more cells are plant cells.

[0207]

[93] According to the method described in

[85] -

[89] , one or more cells are microorganisms, preferably bacterial or fungal cells, such as yeast.

[0208]

[94] The plant according to [7]-[9] is characterized in that the plant further comprises a nucleic acid molecule encoding a polypeptide capable of conveying resistance to the pathogen Beet Necrotic YellowVein Virus (BNYVV) in the plant expressing the polypeptide, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from the following:

[0209] a) A nucleotide sequence encoding a polypeptide having an amino acid sequence according to SEQ ID NO:227 or SEQ ID NO:228,

[0210] b) A nucleotide sequence containing the coding sequence of the DNA sequence according to SEQ ID NO:226.

[0211] c) A nucleotide sequence that hybridizes under stringent conditions with a complementary sequence to a nucleotide sequence according to a) or b).

[0212] d) A nucleotide sequence encoding a polypeptide derived from a polypeptide encoded by the nucleotide sequence of nucleotide sequence according to a) or b) by substitution, deletion and / or addition of one or more amino acids of the amino acid sequence encoded by the nucleotide sequence according to a) or b).

[0213] e) A nucleotide sequence encoding a polypeptide having at least 90%, preferably at least 95%, and most preferably 99% identical amino acid sequences to those encoded by the nucleotide sequence according to a) or b), or

[0214] f) Encoding the following nucleotide sequences: at least one nucleotide-binding domain (NBS) corresponding to amino acid positions 168-227 of SEQ ID NO:227 or amino acid positions 182-241 of SEQ ID NO:228; at least one leucine-rich domain (LRR) corresponding to amino acid positions 591-613 of SEQ ID NO:227 or amino acid positions 605-627 of SEQ ID NO:228; and / or at least one internal repeating domain (IR) corresponding to amino acid positions 1013-1072 of SEQ ID NO:227 or amino acid positions 1027-1086 of SEQ ID NO:228.

[0215]

[95] The plant described in [7]-[9], wherein the plant can be obtained from seeds of the NCIMB deposited in Aberdeen, UK with accession number NCIMB 43646.

[0216]

[96] Seeds or offspring as described in

[10] , wherein the seeds or offspring are available from the NCIMB collection in Aberdeen, UK, accession number NCIMB 43646.

[0217]

[97] The plant according to [7]-[9],

[94] and

[95] is further characterized in that the plant contains SEQ ID NO.182, or can be detected by the marker s4p8772s01.

[0218]

[98] A procedure for breeding sugar beet species resistant to Cercospora, the procedure comprising the following steps:

[0219] i) Obtain the seed or a descendant of the NCIMB deposited in Aberdeen, UK with accession number NCIMB 43646.

[0220] ii) Cultivate the seeds from step i) under conditions that allow for plant growth.

[0221] iii) Hybridize the plants produced in step ii) with sugar beet species.

[0222] The method may optionally include the following additional step:

[0223] iv) Determine the presence or absence of the nucleic acid molecule or nucleotide sequence as described in [1] in the progeny produced by step iii) by the method or procedure described herein, such as the method or procedure described in

[48] -

[71] .

[0224]

[99] Use of electronically transferable and / or electronically storable data derived from the procedures according to any one of

[49] ,

[50] ,

[69] ,

[70] and

[104] -

[113] for determining estimated breeding values ​​of plants in a plant population.

[0225]

[100] According to the use described in

[99] , wherein the estimated breeding value depends at least in part on Cercospora resistance, and wherein the plant population comprises 1,000 or fewer plants.

[0226]

[101] The seed or offspring of a plant according to any one of [7]-[9] or a plant according to

[10] , wherein the nucleic acid molecule or nucleotide sequence according to [1] constitutes or is integrated as an infiltrator.

[0227]

[102] A storage organ of a plant according to any one of [7]-[9] or

[101] . The storage organ may be a taproot, especially the main body of a beet.

[0228]

[103] One or more leaves of a plant according to any one of [7]-[9] or

[101] , wherein the plant may be a genus of Swiss chard or spinach.

[0229]

[104] A method for identifying plants resistant to or tolerant to the genus *Cercospora*, characterized in that the method comprises the following steps:

[0230] (i) Detect the presence or absence of nucleotide sequences selected from the following:

[0231] (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 3;

[0232] (b) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 2;

[0233] (c) A nucleotide sequence comprising a DNA sequence according to SEQ ID No. 1 or SEQ ID No. 53;

[0234] (d) The complementary sequence of the nucleotide sequence according to (a), (b), or (c) is hybridized in 4×SSC (saline-sodium citrate) at 65°C, and then the nucleotide sequence is washed repeatedly in 0.1×SSC at 65°C for a total of about 1 hour.

[0235] (e) A nucleotide sequence encoding a polypeptide having at least 90% identical amino acid sequence to that of the amino acid sequence according to SEQ ID No. 3;

[0236] (f) A nucleotide sequence that is at least 90% identical to the DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2;

[0237] (ii) Detecting the presence or absence of a polypeptide encoded by a nucleotide sequence as defined in step (i) in a plant or a portion of a plant; and / or

[0238] (iii) Detect at least one marker locus in the nucleotide sequence or co-segregating region as defined in step (1),

[0239] The cosegregating region is a genomic region that cosegregates with the Cercospora resistance conferred by the polypeptide or with a nucleic acid molecule or nucleotide sequence, and the cosegregating region contains and is flanked by the markers s4p1395s01 and s4p0421s01.

[0240]

[105] The method according to

[104] is characterized in that the method includes the following additional steps:

[0241] (iv) Select for plants resistant to the genus Cercospora.

[0242]

[106] The method according to

[104] or

[105] , wherein the detection in step (i) or (iii) is based on at least one polymorphism or single nucleotide polymorphism.

[0243]

[107] The method according to

[106] is further characterized in that,

[0244] a) The at least one polymorphism or single nucleotide polymorphism is genetically linked to the nucleotide sequence, or has a recombination frequency of 10% or less with the nucleotide sequence.

[0245] b) The at least one polymorphism or single nucleotide polymorphism is located within 2562 kbp, 2300 kbp, 2100 kbp, 1900 kbp, 1700 kbp, 1500 kbp, 1300 kbp, 1100 kbp, 900 kbp, 700 kbp, 500 kbp, 300 kbp, 100 kbp, 50 kbp, 25 kbp, 10 kbp, 5 kbp, or 1 kbp or less of the nucleotide sequence.

[0246] c) At least one of the polymorphisms or single nucleotide polymorphisms can be detected in seeds deposited in NCIMB at Aberdeen, UK with accession number NCIMB 43646, or

[0247] d) At least one polymorphism or single nucleotide polymorphism is part of a co-segregating region.

[0248] The nucleotide sequence is the nucleotide sequence defined in step (i) of

[104] , and the co-separated region is the co-separated region defined in step (iii) of

[104] .

[0249]

[108] The method according to any one of

[104] -

[107] , wherein step (i) and / or step (iii) further comprises the following step and ends with detection:

[0250] a) Provide at least one plant, its tissue, seed, or at least one cell thereof, and

[0251] b) Extract DNA from at least one plant, its tissue, seed or at least one cell thereof, preferably genomic DNA.

[0252] This means, for example:

[0253] A method for identifying plants resistant to or tolerant of Cercospora, characterized by the following steps:

[0254] ia) provides at least one plant, its tissue, seed, or at least one cell thereof, and

[0255] ib) Extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed, or at least one cell thereof, and

[0256] ic) detects the presence or absence of the nucleotide sequence as defined in step (i) of

[104] ,

[0257] ii) Detecting the presence or absence in a plant or part of a plant of a polypeptide encoded by a nucleotide sequence as defined in step (i); and / or

[0258] iiia) providing at least one plant, its tissue, seed, or at least one cell thereof, and iiib) extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed, or at least one cell thereof.

[0259] (iiic) Detection of at least one marker locus in the nucleotide sequence or co-segregating region as defined in step (1),

[0260] The cosegregating region is a genomic region that cosegregates with the Cercospora resistance conferred by the polypeptide or with a nucleic acid molecule or nucleotide sequence, and the cosegregating region contains and is flanked by the markers s4p1395s01 and s4p0421s01.

[0261]

[109] The method according to

[104] -

[108] involves the use of at least two oligonucleotides.

[0262]

[110] According to the method of

[109] , the oligonucleotide is suitable for use as a primer in PCR and is capable of hybridizing with a genomic spacer region containing and flanked by markers s4p1395s01 and s4p0421s01 or a marker pair as disclosed in

[48] . Preferably, the oligonucleotide can hybridize during PCR with a genomic template containing a nucleotide sequence as defined in step (i) of

[104] in such a manner / distance that the resulting amplification product contains up to 2000 bp, preferably up to 1500 bp, more preferably 1000 bp, and most preferably up to 500 or 200 or up to 100 bp.

[0263]

[111] The method according to any one of the preceding claims, wherein the plant is a plant of the genera *Begonia*, *Spinach*, *Glycine*, *Daucus*, or *Pastinaca*.

[0264]

[112] The method according to any one of

[104] -

[111] involves PCR in steps (i) and / or (iii), wherein the PCR involves two allele-specific forward primers, and wherein the detection step involves fluorescence resonance energy transfer, and wherein the presence, absence, or type of fluorescence is determined by a sensor, wherein a sensor signal is optionally generated. The sensor signal can be converted into electronically transferable and / or electronically storable data representing the detection of the presence of a nucleic acid molecule or nucleotide sequence according to [1]. Furthermore, the electronically transferable and / or electronically storable data can be stored on a computer-readable medium. The sensor's measurement of fluorescence can be an endpoint fluorescence reading.

[0265]

[113] The method according to

[112] also relates to a universal reverse primer. Preferably, the reverse primer and the allele-specific forward primer are hybridized during PCR to a genomic template containing a nucleotide sequence as defined in step (i) of

[104] in such a manner / distance that the resulting amplification product contains up to 2000 bp, preferably up to 1500 bp, more preferably 1000 bp, and most preferably up to 500, 200, or 100 bp. The primer may be an oligonucleotide, such as the oligonucleotide mentioned in

[109] .

[0266]

[114] A plant of the genera *Betula*, *Spinach*, *Soybean*, *Carotene*, or *Papaveris*, comprising a nucleic acid molecule encoding a polypeptide that confers resistance to *Cercospora* in plants expressing the polypeptide, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from the following:

[0267] (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 3;

[0268] (b) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 2;

[0269] (c) A nucleotide sequence comprising a DNA sequence according to SEQ ID No. 1 or SEQ ID No. 53;

[0270] (d) Hybridize the complementary sequence of the nucleotide sequence according to (a), (b), or (c) in 4×SSC at 65°C, and then wash the nucleotide sequence repeatedly in 0.1×SSC at 65°C for a total of about 1 hour.

[0271] (e) A nucleotide sequence encoding a polypeptide having at least 90% identical amino acid sequence to that of the amino acid sequence according to SEQ ID No. 3;

[0272] (f) A nucleotide sequence that is at least 90% identical to the DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2;

[0273] Plants in the genera *Begonia* or *Spinach* also contain an endogenous allele encoding an epsp synthase with an amino acid different from proline at position 179. According to one specific implementation, this epsp synthase is a mutant epsp synthase, and the mutation is generated through mutagenic agents, radiation, gene editing, site-specific point mutations, zinc finger nucleases, TALENS, or by culturing the corresponding cells in vitro and subsequently selecting them using a herbicide (such as glyphosate).

[0274]

[115] The plant described in

[114] is further characterized in that it is a hybrid plant.

[0275]

[116] The plant described in

[114] or

[115] is further characterized in that the plant is a double haploid plant.

[0276]

[117] The plant according to any one of

[114] to

[116] is further characterized in that the resistance to Cercospora is dominant.

[0277]

[118] The plant according to any one of

[114] to

[117] is further characterized in that it is composed of nucleic acid molecules or nucleotide sequences as infiltrates.

[0278]

[119] A plant according to any one of

[114] to

[118] is further characterized in that the nucleic acid molecule or nucleotide sequence is homozygous.

[0279]

[120] The plant according to any one of

[114] to

[119] is further characterized in that the plant has tolerance to glyphosate.

[0280]

[121] The plant according to any one of

[114] to

[120] is further characterized in that a nucleic acid molecule or nucleotide sequence is included as a transgene.

[0281]

[122] The plant according to any one of

[114] to

[121] is further characterized in that the nucleic acid molecule or nucleotide sequence can be obtained from the seeds of NCIMB deposited in Aberdeen, UK with accession number NCIMB 43646.

[0282]

[123] The storage organ or leaf of a plant according to any one of

[114] to

[122] .

[0283]

[124] A granulated seed of a plant according to any one of

[114] to

[122] , characterized in that the seed contains a nucleic acid molecule and an endogenous allele.

[0284]

[125] The plant according to any one of

[114] to

[122] , the storage organ or leaf according to claim 13, or the granulated seed according to claim 14, wherein the epsp synthase having an amino acid different from proline at position 179 comprises an amino acid sequence selected from:

[0285] i) Sequence of SEQ ID NO:223

[0286] ii) Having an amino acid sequence at position 179 that is different from serine and proline, or

[0287] iii) A sequence that is at least 90% identical in length to the sequence in i) or ii).

[0288]

[126] The method according to any one of

[104] -

[113] , wherein the co-separation region is the co-separation region as defined in

[48] .

[0289]

[127] The method according to any one of

[109] -

[113] , wherein at least two oligonucleotides are oligonucleotides as defined in

[20] ,

[21] ,

[72] or

[73] .

[0290]

[128] A mixture of granulated clumps and sugar beet seeds, characterized in that the sugar beet seeds are the seeds of the plant according to any one of

[114] to

[122] , characterized in that the seeds contain nucleic acid molecules and endogenous alleles.

[0291] First, the following explains in detail some of the terms used in this application:

[0292] In the context of this invention, "grading score" should be understood as a qualitative assessment of resistance to Cercospora infection, expressed using a scale from 1 to 9 (where 1 = strong resistance and 9 = no resistance).

[0293] Table 1A: Nine levels of resistance in the genus *Cercospora*

[0294]

[0295] The genus *Cercospora* encompasses a wide variety of species, including, for example, *Cercospora arachidicola*, *Cercospora ariminiensis*, *Cercospora asparagi*, *Cercospora bertoreae*, *Cercospora beticola*, *Cercospora bizzozeriana*, *Cercospora canescens*, *Cercospora carotae*, *Cercospora cistinearum*, *Cercospora cladosporioides*, *Cercospora diazu*, *Cercospora dulcamarae*, *Cercospora erysimi*, *Cercospora hayii*, *Cercospora kikuchii*, and *Cercospora*. malvacearum, Cercospora malvicola, Cercospora medicaginis, Cercospora oryzaem, Cercospora personata, Cercospora plantaginis, Cercospora ricinella, Cercospora setariae, Cercospora unamunoi, Cercospora violae, or Cercospora zeae-maydis.

[0296] The term “approximately” in conjunction with the length of a nucleotide sequence means a deviation of + / - 200 base pairs, preferably + / - 100 base pairs, and particularly preferably + / - 50 base pairs.

[0297] A cosegregating region is a genomic region that, in most cases or always, is inherited along with a genomic region, locus, gene, polymorphism, or single nucleotide polymorphism, and is genetically coupled, genetically linked, or adjacent to or close to the genomic region, locus, gene, polymorphism, or single nucleotide polymorphism. For example, genetic linkage can be inherited to the next generation with at least 95%, 96%, 97%, 98%, or 99%, or the cosegregating region and the genomic region, locus, gene, polymorphism, or single nucleotide polymorphism that are genetically coupled, genetically linked, or adjacent to or close to it have a distance of up to 2562 kbp, 2300 kbp, 2100 kbp, 1900 kbp, 1700 kbp, 1500 kbp, 1300 kbp, 1100 kbp, 900 kbp, 700 kbp, 500 kbp, 300 kbp, 100 kbp, 50 kbp, 25 kbp, 10 kbp, 5 kbp, or 1 kbp or less. The presence of cosegregating regions indicates or diagnoses corresponding genomic regions, loci, genes, polymorphisms, or single nucleotide polymorphisms. Cosegregating regions can be detected / identified, for example, in seeds deposited in NCIMB 43646 in Aberdeen, UK, using the appropriate methods, markers, and oligonucleotides included herein. In the context of this invention, cosegregating regions may be particularly found in the genera *Betula*, *Spinach*, *Glycine*, *Carotene*, or... European windproof Genome regions in plants.

[0298] "Plants" includes plants from different genera, including but not limited to the genera *Begonia*, *Spinach*, *Soybean*, *Carrot*, and *Parsnip*.

[0299] The genus *Betula* belongs to the family Amaranthaceae. These plants include species such as *Betamacrocarpa*, cultivated sugar beet, *Betula lomatogona*, *Betula macrorhiza*, wild white-flowered beet (*Betula corolliflora*), *Betula trigyna*, and *Betula nana*. Cultivated sugar beet species are particularly prevalent, especially the red beet subspecies. Examples include *Betula vulgaris* subsp. *vulgaris* var. *altissima* (the sugar beet in the narrow sense), *Betula vulgaris* ssp. *vulgaris* var. *vulgaris* (Sauvignon Blanc), *Betula vulgaris* ssp. *vulgaris* var. *conditiva* (beetroot / red beet), and *Betula vulgaris* ssp. *vulgaris* var. *crassa* / alba (forage beet). It should be noted that the nucleic acids according to the present invention are not naturally present in beets, chard, beet roots, or fodder beets, but can be introduced into these through human activity.

[0300] Plants of the genus "spinach" belong to the family Amaranthaceae. This genus specifically includes spinach, also known as spinach.

[0301] Plants of the genus "Glycine" belong to the family Fabaceae. This genus particularly includes Glycine max, also known as soybean.

[0302] The genus *Daucus carota* belongs to the family Apiaceae. This genus particularly includes wild carrot (*Daucus carota*) and yellow carrot (*Daucus carota subsp. sativus*), also known as carrot.

[0303] “ European windproof The plant belongs to the Apiaceae family. This genus particularly includes the European parsnip (Pastinaca sativa), also known as the European parsnip.

[0304] The term "functional fragment" in a nucleotide sequence refers to a segment of nucleotide sequence whose function is identical or equivalent to the complete nucleotide sequence from which the functional fragment originates. Therefore, a functional fragment can have nucleotide sequences that are identical or homologous to the total nucleotide sequence in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99% of their length. This explicitly covers the 90-100% range. Furthermore, the term "functional fragment" in a nucleotide sequence can also refer to a segment of nucleotide sequence that, for example, modifies the entire nucleotide sequence post-transcriptionally or during transcriptional gene silencing. Therefore, the functional fragment of the nucleotide sequence may comprise at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the total nucleotide sequence, preferably at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, or 140, and particularly preferably at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 consecutive nucleotides. This also explicitly covers the range of 21 to 50 nucleotides.

[0305] The “functional portion” of a protein refers to a segment of the protein chain or a portion of the amino acid sequence encoding the protein, wherein the segment performs the same or equivalent function as the intact protein in a plant cell. For at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99% of its length, the functional portion of a protein has the same or similar amino acid sequence (taking into account conserved and semi-conserved amino acid exchanges) as the protein from which the functional portion originates.

[0306] The term "heterologous" refers to an introduced polynucleotide that originates from cells or organisms of different genetic backgrounds, of the same or different species, or that is homologous to a prokaryotic or eukaryotic host cell but subsequently located in a different genetic environment, thus differing from the corresponding naturally occurring polynucleotide. Heterologous polynucleotides may exist in addition to the corresponding endogenous gene.

[0307] For the purposes of this invention, "homologous" should be understood as a protein of the same phylogenetic origin; "analogous" should be understood as a protein that performs the same function but has a different phylogenetic origin; "orthologous" should be understood as a protein that performs the same function from a different species; and "paralogous" should be understood as a protein that appears within a species due to replication, wherein such copy retains the same protein function, alters its expression template but does not alter its function, alters its protein function, or divides up the original gene function between two copies.

[0308] "Hybridizing" or "hybridization" should be understood as the process in which a single-stranded nucleic acid molecule binds to a nucleic acid strand that is as complementary as possible, i.e., forms a base pair with it. Standard methods of hybridization are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. It should therefore be understood that at least 60%, more preferably at least 65%, 70%, 75%, 80%, or 85%, and particularly preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases of the nucleic acid molecule form base pairs with the nucleic acid strand that is as complementary as possible. The likelihood of this annealing depends on the stringency of the hybridization conditions. The term "stringency" relates to the hybridization conditions. High stringency exists when base pairing is more difficult; low stringency exists when base pairing is easier. For example, the stringency of hybridization conditions depends on salt concentration or ionic strength and temperature. Generally, stringency can be increased by raising the temperature and / or lowering the salt concentration. "Stringent hybridization conditions" should be understood as those conditions given where hybridization occurs primarily only between homologous nucleic acid molecules. The term "hybridization conditions" thus refers not only to conditions prevalent in the actual addition of nucleic acids but also to conditions prevalent in subsequent washing steps. For example, stringent hybridization conditions are those under which nucleic acid molecules with at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity hybridize primarily. A stringent hybridization condition is, for example, hybridization at 65°C in 4×SSC followed by repeated washing at 65°C in 0.1×SSC for a total of approximately 1 hour. Hybridization preferably occurs under stringent conditions.

[0309] A sequence that shows a specific level of identity with the starting sequence can show that given level of identity across the entire length of the starting sequence. This applies to both nucleotide sequences and amino acid sequences.

[0310] For nucleic acids in the form of double-stranded DNA, a "complementary" nucleotide sequence means that, according to the base pairing rule, the second DNA strand, complementary to the first DNA strand, has nucleotides corresponding to the bases of the first strand. The complementary sequence is preferably perfectly complementary to the reverse sequence and therefore preferably has the same length.

[0311] "Isolated nucleic acid molecules" should be understood as nucleic acid molecules extracted from their natural or pristine environment. This term also encompasses synthetically produced nucleic acid molecules. "Isolated polypeptides" should be understood as polypeptides extracted from their natural or pristine environment. This term also encompasses synthetically produced polypeptides.

[0312] "Molecular marker" or "marker" refers to a polymorphic nucleic acid in a plant population and serves as a reference or orientation point. Depending on the technical context, the term "marker" can refer to a specific genomic location detectable by a corresponding "molecular marker," where the "molecular marker" is compatible with the genomic location sequence in most cases. Markers used to detect recombination events should be suitable for monitoring differences or polymorphisms within a plant population. Thus, such markers are capable of detecting and distinguishing various allelic states (alleles). The term "molecular marker" also refers to nucleotide sequences that are complementary to or at least largely complementary to or homologous to the genomic sequence, such as nucleic acids used as probes or primers. These differences at the DNA level can be found as markers and are, for example, polynucleotide sequence differences such as SSR (simple sequence repeat), RFLP (restriction fragment length polymorphism), FLP (fragment length polymorphism), or SNP (single nucleotide polymorphism). Markers can be derived from genomic or expressed nucleic acids, such as spliced ​​RNA, cDNA, or ESTs, and may also be associated with nucleic acids used as probes or primer pairs, thus suitable for amplifying sequence fragments using PCR-based methods. Markers describing genetic polymorphism (across different parts of a population) can be detected using well-established methods in the prior art (An Introduction to Genetic Analysis, 7th Edition, Griffiths, Miller, Suzuki et al., 2000). These include, for example, DNA sequencing, PCR-based sequence-specific amplification, validation of RFLP, validation of polynucleotide polymorphisms using allele-specific hybridization (ASH), detection of amplified variable sequences in plant genomes, detection of 3SRs (self-persistent sequence replication), and detection of SSRs, SNPs, RFLPs, or AFLPs (amplified fragment length polymorphisms). Furthermore, methods for detecting ESTs (expressed sequence tags) and SSR markers derived from EST sequences and RAPDs (randomly amplified polymorphic DNA) are also known. Depending on the context, the term "marker" in this specification may also refer to a specific chromosomal location in the genome of a species in which a particular marker (e.g., an SNP) can be found.

[0313] The label also includes a synthetic oligonucleotide that can be linked to one or more detection molecules, wherein, within the scope of the validation method, the detection molecules can be used to detect the generation of a reaction or signal. The synthetic oligonucleotide also includes a labeled primer. Both the synthetic oligonucleotide and the labeled primer are artificial compounds that do not exist in nature and cannot be isolated from nature. The generation of such compounds is explained further below.

[0314] A “single nucleotide polymorphism” or SNP is a genetic variation between two DNA samples in which at least one nucleotide differs between the two samples. In most cases, the samples belong to two of the same species, and the comparison or alignment of the two samples is performed based on homologous genomic regions. SNPs may cause allelic variations, but not all SNPs need to be present within functional genomic elements such as genes. SNPs can be used, for example, to distinguish between different genotypes / haplotypes, or to screen and select for the presence or absence of functional genomic elements (e.g., specific genes or their allelic variants). Due to genetic linkage, SNPs do not need to be within the functional genomic element to be selected. Examples of SNPs in the sense of this invention are given in Table 1b, especially those contained in universal primer sequences. The detection or identification of SNPs can be performed by PCR involving two different forward primers and a universal reverse primer. Technical details of such detection or identification can be obtained from the above paragraphs

[67] -

[70] .

[0315] A promoter is a non-translated regulatory DNA sequence, typically upstream of a coding region, that contains the binding site of RNA polymerase and initiates transcription of the DNA. Promoters additionally contain other elements that act as regulators of gene expression (e.g., cis-regulatory elements). A core or minimal promoter is a promoter that contains the essential elements required to initiate transcription (e.g., a TATA box and / or an initiator).

[0316] "Pathogen" refers to an organism that interacts with a plant and causes disease symptoms in one or more organs of the plant. Examples of pathogens include animals, fungi, bacteria, viruses, or oomycetes.

[0317] “Pathogenic infection” should be understood as the earliest point in time when a pathogen interacts with the plant host tissue. In this sense, “infection” refers to the contact that occurs between the pathogen and the host. As the pathogen anchors itself on the host (e.g., fungal spores on the surface of plant leaves), mechanisms for pathogen detection and signal transduction begin in the plant host cells. In the case of *Cercospora beta*, conidia form under humid, warm weather and are transferred to neighboring plants by rain and wind. New infections most often first appear as single leaf spots on physiologically older outer leaves. These are most frequently separated from healthy leaf tissue by a fairly clear brown ring. The brown conidial carriers of the fungus can be observed with the aid of a magnifying glass in the center of the spot (grade 3). The number of these brown spots increases rapidly, with the sporocarps initially overlapping even smaller necrotic areas (grade 5). In further progression (now also extending to the inner leaves), final necrosis occurs first on the outer leaves (grade 7), followed by necrosis of almost all leaves (grade 9). The course and severity of symptoms are strongly dependent on location and fluctuating weather conditions from year to year.

[0318] Plant “organs” refer to, for example, leaves, shoots, stems, roots, hypocotyls, vegetative buds, meristems, embryos, anthers, ovules, seeds, or fruits. “Plant parts” include, but are not limited to, shoots or stalks, leaves, flowers, inflorescences, roots, fruits, and seeds, as well as pollen. The term “plant part” also refers to a combination of multiple organs, such as a flower or seed, or a portion of an organ, such as a cross-section running through a young plant shoot. Plant “tissues” include, for example, callus, storage tissue, meristems, leaf tissue, bud tissue, root tissue, plant nodule tissue, or reproductive tissue, as well as new tissue, parenchyma tissue, vascular tissue, sclerenchyma tissue, and epidermis. However, tissues are not limited to those listed. For example, a plant “cell” should be understood as, for example, a separate cell or aggregate of cells with a cell wall, or a protoplast.

[0319] "Recombination frequency" refers to the frequency of recombination that occurs during meiosis between genomic elements such as single nucleotide polymorphisms, genes, loci, QTLs, or genetic regions. A 1% recombination frequency equals 1 cM. Recombination frequencies can be determined by generating meiotic maps.

[0320] "The resistance gene according to the invention" is a nucleotide molecule or nucleic acid sequence as described below paragraph [1]. The gene may be genetically linked to a cosegregating region.

[0321] "Variety" refers to a group of plants within a single, known lowest-level taxonomic group, regardless of whether it fully meets the conditions for granting breeder's rights.

[0322] - Defined by the expression of traits produced by a given genotype or combination of genotypes.

[0323] -Distinguishing it from any other plant group by the expression of at least one of the aforementioned characteristics, and

[0324] - It is considered a unit in terms of its unchanging reproductive fitness.

[0325] In conjunction with this invention, the term "regulatory sequence" refers to a nucleotide sequence that affects specificity and / or expression intensity, for example, because the regulatory sequence confers defined tissue specificity. For example, such a regulatory sequence may be located upstream or downstream of the transcription start site of a minimal promoter, either in a transcribed but untranslated leader sequence or within an intron.

[0326] The term "resistance" should be interpreted broadly and encompasses a range of protection, from delaying to completely halting disease progression. An important example of a pathogen is *Cercospora betaine*. The resistant plant cells or resistant plants of the present invention preferably achieve resistance to *Cercospora betaine*. Resistance to a pathogen is equivalent to resistance to a disease caused by that pathogen; for example, resistance to *Cercospora betaine* is also resistance to leaf spot. For example, an increase in resistance can be measured by a reduction in fungal biomass on the host plant; for this purpose, fungal DNA can be determined by comparing it with plant DNA in infected plant tissue using quantitative PCR. An additional method for measuring resistance is optical grading, where grades are assigned from 1 (uncompetent) to 9 (very susceptible).

[0327] "Transgenic plant" refers to a plant whose genome incorporates at least one polynucleotide. This can be a heterologous polynucleotide. Preferably, the polynucleotide is stably integrated, meaning that the integrated polynucleotide is stably preserved in the plant, expressed, and can be stably passed on to offspring. Stable introduction of a polynucleotide into a plant genome also includes integration into the genome of a previous parent plant, where the polynucleotide can be further stably passed on. The term "heterologous" means that the introduced polynucleotide originates from cells or organisms of different genetic backgrounds, of the same or different species, or is homologous to, for example, prokaryotic or eukaryotic host cells, but subsequently located in a different genetic environment and therefore differs from the corresponding polynucleotide that may exist naturally. Heterologous polynucleotides may also exist in addition to the corresponding endogenous gene.

[0328] "Raw material for industrial sugar production" refers to plant material that can be fed into specialized sugar-refining equipment for extracting sugar from sugar beets. Such raw material is typically the main beet root (taper) of harvested sugar beets. To ensure consistency with the extraction process, the beet root needs to have sufficient mass, volume, and conical shape to allow for mechanical cutting into strips (beet strips). These beet strips maximize the surface area for sugar extraction and should have low sodium, potassium, and nitrogen content to allow for efficient extraction. After extraction, the remaining beet pulp is pressed, dried, and used as animal feed.

[0329] "Sucrose concentration" is expressed as a percentage of the fresh weight of the root.

[0330] "Monoembryo" means that a seed grows completely into a single plant, while a polyembryo or polyembryonic seed (also called a "bulb") grows into several plants.

[0331] Bolting is the process by which sugar beets produce a single (or multiple) flower stalk in an attempt to naturally produce seeds and reproduce. Bolting in sugar beets is triggered by vernalization, such as low-temperature stress that may occur during winter. However, commercially grown sugar beets are harvested before bolting because the bolting process and subsequent fruit setting reduce the sucrose content in the beet mass.

[0332] "Introgression" means that a nucleotide sequence has been transferred into the genome of a plant from a plant that does not belong to the same species or subspecies. For example, this could mean that a nucleotide sequence originating from the coastal beet subspecies has been transferred to the red beet subspecies.

[0333] The design and embodiments of the invention are described by way of example with reference to the accompanying sequence and figures.

[0334] Figure 1 : Protein sequence alignment between resistance proteins (proteins that confer resistance to the genus *Cercospora*) and sensitivity proteins (proteins that do not confer resistance to the genus *Cercospora*). Polymorphisms are highlighted in gray.

[0335] Figure 2 The genetic (left) and physical (right) map of the genome region containing the resistance gene shows the locations of some of the most important markers.

[0336] Figure 3 : Vector map of pZFN-nptII, including the LRR region.

[0337] Figure 4 : Evaluation of statistical cassette plots of data generated eight days after infection during transgenic validation of the resistance gene.

[0338] Figure 5: Evaluation of statistical cassette plots of data generated eleven days after infection during transgenic validation of the resistance gene.

[0339] Figure 6 : Evaluation of statistical cassette plots of data generated eight days after infection during transgenic validation of the resistance gene.

[0340] Figure 7 : Evaluation of statistical cassette plots of data generated 15 days after infection during transgenic validation of the resistance gene. Detailed Implementation

[0341] This invention relates to a nucleic acid molecule capable of conferring resistance to *Cercospora* in plants (particularly red beets) expressing polypeptides encoded by the molecule. According to a preferred embodiment of the invention, the pathogen is the fungus *Cercospora betaine*, one of the most important and destructive leaf pathogens in sugar beets, beet roots, and chard, causing crop losses exceeding 40%. This fungus produces a secondary metabolite, cercosporacin, which reacts with oxygen in the presence of light, leading to the formation of reactive oxygen species (ROS). ROS cause extensive cell damage in the leaf tissues of infected plants, manifesting as necrosis.

[0342] This invention is based on the fine mapping, identification, isolation, and characterization of genes or loci derived from the donor coastal sugar beet, whose presence in plants (particularly red sugar beet) is associated with, or is the cause of, plant resistance to Cercospora leaf spot. The initial material was a coastal sugar beet population cultivated from 37 coastal sugar beet accessions from different sources. Seeds of red sugar beet plants (sugar beet cultivar 6626) containing the resistance according to the invention were deposited with accession number NCIMB 43646 prior to submission to the National Collection of Industrial Food and Marine Bacteria (NCIMB), Aberdeen, UK. In particular, sugar beet plants, sugar beet cultivars, or red sugar beet subspecies containing the resistance gene according to the invention can be derived from the deposited seeds.

[0343] The nucleotide and amino acid coding sequences of the nucleic acid molecules according to the present invention are characterized by a variety of polymorphisms, which distinguish the NPS-LRR gene identified according to the present invention from "sensitive" variants of the gene (i.e., gene variants that do not confer resistance to Cercospora). Examples of polymorphism include... Figure 1 As shown.

[0344] The nucleic acid molecule according to the invention can be an isolated nucleic acid molecule. It is preferably DNA, and particularly preferably cDNA (encoding DNA). The polypeptide encoded by the nucleic acid molecule according to the invention preferably confers resistance to *Cercospora betaine*, the pathogen causing the plant disease *Cercospora* leaf spot. Furthermore, the polypeptide encoded by the nucleic acid molecule according to the invention confers—in particular—resistance to the *Betula* genus. The plant is preferably a sugar beet cultivar, particularly preferably a red beet subspecies; for example, these include cultivars of sugar beet, beetroot, forage beet, chard, and Swiss chard.

[0345] In one embodiment of the invention, the nucleic acid molecule according to the invention comprises the following nucleotide sequence: a polypeptide encoding an amino acid sequence according to SEQ ID No. 3, and / or a DNA sequence encoding a sequence according to SEQ ID No. 2. Furthermore, the invention provides nucleotide sequences comprising DNA sequences according to SEQ ID No. 1 and SEQ ID No. 53.

[0346] The gene identified according to the present invention is an NBS-LRR type resistance gene / protein characterized by a specific structural motif. The general structure of such resistance proteins in plants has been well examined (Martin et al., Annual Review Plant Biology 54 (2003), 23-61). However, the principles of the structural implementation scheme (specifically referred to as the LRR domain, used as a potential detection domain for most unknown pathogenic effectors) are unpredictable, and the functional background (i.e., genetic structure) of the resistance gene is often largely unknown. Therefore, it is impossible to identify genes or proteins conferring resistance to Cercospora solely based on known structural motifs. Furthermore, the sequence region has been shown to be a highly repetitive region containing tandem repeats with very high sequence homology, which makes the development of diagnostic markers and the assembly of sequence data particularly difficult.

[0347] By establishing populations of over 4,000 dividing progeny groups and developing specialized recombination screening, the target region was narrowed and thus further segregated through analysis of informative recombinants (genotype and phenotype) in a series of resistance tests. This genetic mapping, along with the creation of a physical map using accompanying WHG sequencing (“whole genome sequencing”), comparative BAC (Bac-by-Bac) sequencing, and bioinformatics analysis, led to the identification of three recombinant genotypes of the resistance gene (one with one recombinant in adjacent genes, and two with two recombinants in adjacent genes). Given the specific requirements, the inventors placed highly repetitive structures within the target region, containing tandem repeat sequences with very high sequence homology, which made marker development and therefore the identification of informative recombinants more difficult. The following steps are particularly crucial for locating the genetic structure of the resistance gene:

[0348] - Development of the tags s4p0264s01, s4p2271s01, sxh0678s01, s4p4293s01, s4p4295s01, and s4p4301s01 (see Table 1B).

[0349] - Fine-tuning is achieved by combining with dense phenotyping. In greenhouse testing, the phenotype is validated using 90-180 offspring per plant and dense statistical methods (e.g., t-tests, power analysis, etc.).

[0350] - Identify and sequence BAC clones from a BAC library of resistant genotypes.

[0351] - Sequence evaluation, and comparison of sequences and proteins between RR (i.e., resistance) and ss (i.e., sensitivity) genotypes; thus, due to sequence complexity, it is not always possible to perform an explicit assembly of RR and ss sequence data.

[0352] Table 1B: Markers in target regions involving susceptible genotypes, resistant genotypes, and shared sequences.

[0353]

[0354]

[0355]

[0356] The compounds provided in Table 1B can be used as molecular markers according to the invention. All markers are suitable for detecting genetic material containing resistance genes according to the invention (especially by identifying genetically linked polymorphisms such as single nucleotide polymorphisms) and for tracing the inheritance of genetic material from source plant to its offspring via marker-assisted selection (MAS). In this respect, the markers disclosed herein are highly suitable for transferring resistance genes according to the invention from one subspecies to different subspecies. The markers disclosed herein can also be used for genetic mapping and tracing recombination as a result of meiosis. The resistance gene appears to be located near or between physical locations of 57219956 bp and 57243521 bp. The markers disclosed herein located very close to this region are highly suitable for detecting the resistance gene. The markers disclosed herein located further away from the resistance gene still show strong genetic linkage to the resistance gene but can also be used to detect rare recombination events and genetic exchanges in the flanking regions of the resistance gene. This can be used, for example, for backcrossing methods and for the transfer of resistance genes to different genetic backgrounds. The markers disclosed herein, especially those that are far from the resistance gene, are particularly useful when the resistance gene is passed on to offspring as part of a cosegregating region. Resistance genes including cosegregating regions can also be passed from one subspecies to different subspecies. Successful transfer can be confirmed by the markers disclosed herein, for example, by detecting the resistance gene or the cosegregating region. The markers disclosed herein can also be used to reduce the cosegregating region in backcross programs involving several generations, while maintaining the resistance gene according to the invention in offspring. Additional markers for detecting the resistance gene according to the invention can be derived from the data disclosed herein. In particular, those markers located in genomic spacers flanked by markers s4p0421s01 and s4p1395s01 or flanked by the marker pairs given in the above paragraph

[48] can be derived. The genomic material contained in the seeds of NCIMB deposited in Aberdeen, UK with accession number NCIMB43646 is beneficial for the derivation of additional markers. This genomic material allows, for example, comparison of genetic material encoding resistance according to the invention with genetic material lacking the resistance gene according to the invention. The genetic material lacking resistance can be a homolog or located at the same physical position as the genetic material encoding resistance according to the invention (compare Table 1b). These alignments can be used to identify polymorphisms, such as single nucleotide polymorphisms. Based on polymorphisms, additional markers can be created to detect the identified polymorphisms, especially those genetically linked to the resistance gene according to the invention. Alignment and detection of polymorphisms based on alignments typically involve the use of a computer and the storage of data on a computer-readable medium. It is also proposed that signals generated by molecular markers during genetic material analysis be converted into electronically transferable and / or electronically storable data, which can be processed by means of a computer. Such computer-based procedures can involve determining or calculating estimated breeding values ​​for plants.Estimated breeding values ​​can cover levels of resistance or tolerance to the genus *Cercospora*. More information on breeding value estimation is available in US8321147B2.

[0357] Analysis revealed that the LRR gene shares moderate protein homology (sequence identity 322 / 830 = 38%) with the Cf-2 resistance protein from tomato (UNIPROT|Q41397_SOLPI P.Cf-2.1). In fact, the identified protein conferring resistance to *Cercospora* is the best sugar beet protein homolog of the Cf-2 tomato resistance protein. The Cf-2 resistance protein from tomato confers resistance to *Cladosporium fulvum* (a type of black mold) via interaction with the non-toxic protein Avr2 from *C. fulvum* (US 6,287,865 B1). This leads to the activation of the plant's immune defenses against pathogens; see Dixon et al., 1996 (Dixon, Mark S. et al., "The tomato Cf-2 disease resistance locus comprises two functional genes encoding leucine-rich repeat proteins." Cell 84.3(1996):451-459). Due to the sequence homology between the Cf-2 gene and the identified LRR gene, it can be hypothesized, but is not bound by a single theory, that a similar defense mechanism constituting the basis of Cercospora resistance exists in sugar beets. However, given the moderate sequence homology, different mechanisms cannot be ruled out.

[0358] Furthermore, substitutions, deletions, insertions, additions, and / or any other alterations can be introduced, alone or in combination, into the nucleotide sequence according to the invention. These alterations do indeed change the nucleotide sequence, however, the modified nucleotide sequence performs the same function as the original sequence. This invention relates to the encoding of amino acid sequences conferring resistance to Cercospora leaf spot. Therefore, in another embodiment, the invention includes a nucleotide sequence encoding a polypeptide, which represents a derivative of a polypeptide encoded by the nucleotide sequence according to the invention, or includes an amino acid sequence according to the invention. A derived amino acid sequence having at least one substitution, deletion, insertion, or addition of one or more amino acids represents a derivative of a polypeptide, wherein the functionality of the encoded polypeptide / protein is preserved. Thus, substitutions, deletions, insertions, additions, and / or any other alterations, alone or in combination, can be introduced into the nucleotide sequence using conventional methods known in the art, such as via site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, genome editing, etc., these alterations do indeed change the nucleotide sequence but perform the same function as the original sequence.

[0359] The substitution of an amino acid by a different amino acid having the same or similar chemical / physical properties is called a "conservative substitution" or "semi-conservative substitution." Examples of the physical / chemical properties of an amino acid include hydrophobicity or charge. Which amino acid substitution represents a conservative or semi-conservative substitution is known to those skilled in the art. Furthermore, general expertise allows those skilled in the art to identify, characterize, and detect which amino acid deletions and additions are harmless to the functionality of the antagonistic protein, and at which positions these are possible. Those skilled in the art will understand that, in the case of the NBS-LRR protein of the present invention being used to modify amino acids (substitution, deletion, insertion, or addition of one or more amino acids), the functionality of particularly conserved domains must be preserved, and therefore only a limited number of the aforementioned modifications are possible in these domains.

[0360] The present invention therefore includes functional fragments of nucleotide sequences according to the invention. Thus, the term "fragment" includes a gene having a nucleotide sequence that is sufficiently similar to the aforementioned nucleotide sequence. The term "sufficiently similar" means that the first nucleotide sequence or amino acid sequence has a sufficient or minimum number of identical or equivalent nucleotide or amino acid groups relative to the second nucleotide sequence or second amino acid sequence.

[0361] Regarding the amino acid sequences, after modification by the foregoing methods, they also share common structural domains and / or common functional activities. A nucleotide or amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% similarity to the nucleotide or amino acid sequence according to the invention is defined herein as sufficiently similar. This also explicitly covers the range of 90% to 100%. For functional fragments, sufficient similarity is established if the nucleotide or amino acid sequence generally has the same characteristics as the previously specified nucleotide or amino acid sequences of the invention. Those nucleotide sequences that directly or indirectly (e.g., via amplification or replication steps) generate the encoding derivative or the amino acid sequence for which the derivative is derived from an initial nucleotide sequence that corresponds in its entire length or at least partially to the nucleotide sequence according to the invention.

[0362] Therefore, the present invention includes nucleotide sequences capable of hybridizing under stringent conditions with nucleotide sequences complementary to the nucleotide sequence according to the present invention or the nucleotide sequence encoding the amino acid sequence according to the present invention.

[0363] In another embodiment, the nucleic acid molecule according to the invention is characterized in that, upon expression in a plant, it inherently confers a dominant resistance effect against the pathogen (preferably against *Cercospora beta*), or it encodes a polypeptide capable of conferring a dominant resistance effect against *Cercospora*. In a preferred embodiment, the nucleic acid molecule or the polypeptide confers at least one grade score, preferably at least two grade scores, and particularly preferably three to four grade scores of resistance. Such genes are unknown in the art: they inherently confer this strong and significant resistance to *Cercospora*, or encode a polypeptide capable of conferring such significant resistance. As described above, in previously commercially available varieties, *Cercospora* resistance is transmitted via numerous resistance genes with relatively small effects, and a disadvantage of these varieties is that their development is very slow and expensive due to complex transmission, and their crop yields are significantly lower than normal varieties in the absence of infection. Among other things, this may be related to the epigenetic interaction between some resistance genes and genes responsible for sugar production, which leads to reduced plant fitness in the absence of pathogens.

[0364] Therefore, the inventors have for the first time provided a Cercospora resistance gene that can be used to significantly simplify breeding. By introducing this gene into superior lines, it is now possible to develop very high-yielding varieties with high Cercospora resistance very rapidly. Thus, within the framework of this invention, sugar beet plants, chard plants, red beet or beet root plants, and forage beet plants with resistance to Cercospora beetula according to the invention are provided for the first time, and are therefore covered by this invention. Since the listed plants are all cultivated plants, crops or plants suitable for agricultural cultivation and possessing resistance according to the invention are part of this invention. In particular, crops that contain underground storage organs that can be used as food, raw material, or industrial sources of sugar, and contain resistance according to the invention, constitute another aspect of this invention. The storage organs may be, for example, the sugar beet body of sugar beet, the consumable beet body of red beet, or the feedable beet body of forage beet. The total amount of underground storage organs may be more than 50%, and for sugar beet, even more than 70% of the total mass of a fully developed plant. Furthermore, the seeds or sowing materials of these plants are also part of this invention. The seeds or sowing materials may be technically processed as further described below. Part of this invention also includes plants of the genera *Spinach*, *Soybean*, *Carrot*, and *Paspalum* that contain the resistance gene according to the invention. Spinach species and varieties containing the resistance gene according to the invention are particularly included. The storage organ may be a taproot, especially the main stem of a beet.

[0365] The resistance gene according to the invention can be under the control of or operably linked to the promoter according to SEQ ID No. 7. The resistance gene can also be under the control of or operably linked to a nucleic acid molecule containing the promoter according to SEQ ID No. 7. This aspect covers sequences having 90%, 95%, or 99% sequence identity with SEQ ID No. 7 in full length.

[0366] In this context, the present invention also includes a nucleic acid encoding the protein according to SEQ ID No. 3, wherein, in a particular embodiment, the nucleic acid according to SEQ ID No. 1 is excluded.

[0367] Furthermore, the present invention relates to recombinant and / or heterologous DNA molecules comprising sequences of nucleic acid molecules according to the present invention. Furthermore, the DNA molecule preferably has a regulatory sequence. Thus, it can be operatively linked to or influenced by the regulatory sequence. The regulatory sequence is preferably a promoter sequence and / or other sequences of transcription or translation control elements, such as cis elements. The regulatory sequence controlling the expression of a gene comprising a nucleic acid molecule according to the present invention is preferably a sequence capable of conferring or regulating expression due to pathogen infection. The promoter is preferably capable of controlling the expression of a DNA sequence specifically in plant leaves. The regulatory sequence may be heterologous to the expression sequence. The advantage of this approach is that those skilled in the art can better regulate the expression rate, the tissue in which expression occurs, and the timing of expression by selecting the regulatory sequence most suitable for their respective use cases. The heterologous DNA sequence preferably comprises nucleotide sequences encoding components of plant pathogen defense (e.g., resistance genes (R-genes) or genes encoding enzymes involved in signal transduction, such as kinases or phosphatases, and for G-proteins, or encoding pathogenic effectors (called avirulence genes (avr))). The heterologous DNA sequence may be one of the DNA sequences according to the present invention. Heterologous DNA sequences can also encode additional components of plant pathogen defense. Therefore, heterologous DNA sequences can be programmed to produce polycistronic mRNAs after transcription.

[0368] The present invention also relates to polypeptides encoded by nucleic acid molecules according to the invention, and functional and / or immunologically active fragments thereof, as well as antibodies that specifically bind to the polypeptide or fragments thereof. The polypeptide particularly preferably has an amino acid sequence according to SEQ ID No. 3. Recombinant production of proteins, polypeptides, and fragments is well known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; or Wingfield, PT, 2008, Production of Recombinant Proteins, Current Protocols in Protein Science, 52:5.0:5.0.1–5.0.4). Polyclonal or monoclonal antibodies against proteins according to the invention can be produced by those skilled in the art using known methods (E. Harlow et al., ed., Antibodies: A Laboratory Manual (1988)). The generation of monoclonal antibodies, as well as Fab and F(ab')2 fragments that can also be used in protein detection methods, can be performed via various conventional methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118, New York: Academic Press (1983)). The antibodies can then be used to screen expressed cDNA libraries for the identification of identical, homologous, or heterologous genes via immunological screening (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, or Ausubel et al., 1994, “Current Protocols in Molecular Biology.” John Wiley & Sons), or for Western blot analysis. In particular, the present invention relates to antibodies that selectively detect polypeptides encoded by alleles conferring resistance to Cercospora according to the present invention, and substantially do not detect polypeptides encoded by corresponding sensitive alleles, i.e., they detect polypeptides encoded by corresponding sensitive alleles that are 2 times less, preferably 5 times less, and more preferably 10 times or more less than polypeptides encoded by alleles conferring resistance to Cercospora according to the present invention.

[0369] In a preferred embodiment, the antibody according to the invention is characterized in that it is a synthetic polypeptide that does not exist in nature.

[0370] Furthermore, the antibodies according to the invention can be conjugated with a fluorescent dye for use, for example, in immunohistochemical methods, to induce antibody staining. The fluorescent dye can be a fluorescent pigment. The antibodies according to the invention can also be conjugated with other signaling molecules, including, for example, biotin, radioisotopes, reporter enzymes (such as alkaline phosphatase), or oligonucleotides.

[0371] An additional subject of the invention is a vector or expression cassette comprising a nucleic acid molecule or recombinant DNA molecule according to the invention—which may be under the control of regulatory elements, and particularly functional regulatory elements in plants; and negative and / or positive selection markers. Thus, the vector backbone is heterologous to the nucleic acid molecule according to the invention, meaning that such a vector does not exist in nature and cannot be isolated from nature. The vector is a plasmid, granule, phage, or expression vector, transformation vector, shuttle vector, or cloning vector; it may be double-stranded or single-stranded, linear or circular; or it may be integrated into its genome or transformed extrachromosomally into prokaryotic or eukaryotic organisms. The nucleic acid molecule or DNA molecule according to the invention in the expression vector or expression cassette is preferably operatively linked to one or more regulatory sequences that allow transcription and optional expression in prokaryotic or eukaryotic cells (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). These regulatory sequences are preferably promoters or terminators, particularly transcription initiation sites, ribosome binding sites, RNA processing signals, transcription termination sites, and / or polyadenylation signals. For example, nucleic acid molecules are here under the control of appropriate promoters and / or terminators. Suitable promoters can be constitutive promoters (e.g., the 35S promoter from cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812); pathogen-induced promoters are particularly suitable (e.g., the PR1 promoter from parsley (Rushton et al., EMBO J.15 (1996), 5,690–5,700)). Particularly suitable pathogen-induced promoters are synthetic or chimeric promoters not found in nature, composed of multiple elements, containing a minimal promoter, and having at least one cis-regulatory element upstream of the minimal promoter, where at least one cis-regulatory element serves as the binding site for a specific transcription factor. Chimeric promoters are designed according to desired needs and can be induced or repressed via different factors. Examples of such promoters can be found in WO 00 / 29592, WO2007 / 147395, and WO 2013 / 091612. For example, a suitable terminator is the nos terminator (Depicker et al., J.Mol.Appl.Genet.1(1982), 561-573). Suitable promoters and terminators may also be natural promoters and natural terminators, whose DNA sequences are copied in SEQ ID No.7 and 8.The vector or expression cassette additionally contains conventional indicator / reporter genes or resistance genes for detecting the transfer of the desired vector or DNA / nucleic acid molecule, and for selecting individuals containing the vector or DNA / nucleic acid molecule, since direct detection via gene expression is often quite difficult. Since the nucleic acid molecule according to the invention itself encodes a polypeptide conferring resistance to Cercospora leaf spot, providing an additional resistance gene for expression in plant cells is not necessary; however, this method is recommended to allow for rapid selection.

[0372] Examples of indicator / reporter genes include, for example, luciferase genes and genes encoding green fluorescent protein (GFP). Furthermore, these indicators also allow for the testing of gene promoter activity and / or regulation. In particular, examples of resistance genes used for plant transformation are neomycin phosphotransferase genes, hygromycin phosphotransferase genes, or genes encoding phosphinic acid acetyltransferases. Additional positive selection markers may be enzymes that provide a selection advantage over untransformed plants, particularly a nutritional advantage, such as mannose-6-phosphate isomerase or xylose isomerase. However, this does not preclude additional indicator / reporter genes or resistance genes known to those skilled in the art. In a preferred embodiment, the vector is a plant vector. Furthermore, the expression cassette may be present as a form integrated into the plant genome.

[0373] In another aspect, the present invention relates to cells comprising the vector, recombinant DNA molecule, and / or nucleic acid molecule according to the invention. Cells in the sense of the invention can be prokaryotic (e.g., bacterial) or eukaryotic cells (e.g., plant cells or yeast cells). Cells are preferably Agrobacterium, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, Escherichia coli cells, or plant cells; plant cells are particularly preferably cells of plants of the genus *Saccharum*, sugar beet species, or subspecies *Betula*. Cells may also be present as cultures. Therefore, the invention also covers cell cultures containing such cells. Cell cultures are preferably pure cultures or isolates that do not contain another type of cell.

[0374] Those skilled in the art are aware of various methods, such as conjugation or electroporation, by which nucleic acid molecules, recombinant DNA molecules, and / or vectors or expression cassettes according to the invention can be introduced into Agrobacterium, as well as various transformation methods (biotransformation, Agrobacterium-mediated transformation) by which nucleic acid molecules, DNA molecules, and / or vectors according to the invention can be introduced into plant cells (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0375] Furthermore, the present invention preferably relates to cercariae-resistant plants containing nucleic acid molecules according to the invention that confer resistance to cercariae, preferably plants or parts of the red beet species. These cercariae-resistant plants may contain nucleic acid molecules according to the invention as transgenic or as endogenous organisms. Within the scope of the present invention, red beet subspecies plants containing nucleic acid molecules according to the invention have been produced for the first time. The present invention also includes red beet subspecies plants containing nucleic acid molecules according to the invention as endogenous organisms.

[0376] Thus, a portion may be a cell, tissue, organ, or a combination of multiple cells, tissues, or organs. A combination of multiple organs may be, for example, a flower or a seed. The resistant *Cercospora* plants of the present invention exhibit higher resistance to *Cercospora* (particularly *Cercospora betaine*) than corresponding plants (control plants) that do not contain the nucleic acid molecules according to the present invention. The control plant ideally has the same genotype as the transgenic plant and is cultured under the same conditions, but does not contain the resistance-conferring nucleic acid molecules. The level of resistance of the *Betula* plant, for example, to *Cercospora betaine*, can be qualitatively determined by determining the grading score. Higher resistance is manifested by an improvement in at least one grading score, at least two grading scores, and preferably at least three or more grading scores.

[0377] Compared to corresponding plant cells or plants or parts thereof that do not contain the nucleic acid molecule according to the invention or contain a sensitive allele variant of the nucleic acid molecule, plant cells or plants or parts thereof of the invention containing the nucleic acid molecule according to the invention (especially beet plants) preferably exhibit higher resistance to pathogens (especially to *Cercospora betaecarpa*). The level of resistance of beet plants, for example to *Cercospora betaecarpa*, can be qualitatively determined by determining a grade score. Higher resistance is manifested by an improvement in at least one grade score, at least two grade scores, and preferably at least three or more grade scores.

[0378] In the case of transgenic plant cells, plants, or parts thereof, this comprises nucleic acid molecules or DNA molecules according to the invention as transgenic vectors or expression cassettes of the invention. Such transgenic plant cells, plants, or parts thereof are preferably stably transformed, for example, with nucleic acid molecules, DNA molecules according to the invention, or with vectors or expression cassettes of the invention. In a preferred embodiment, the nucleic acid molecule is operatively linked to one or more regulatory sequences that allow transcription and, optionally, expression in plant cells. The overall structure consisting of the nucleic acid molecule according to the invention and one or more regulatory sequences then represents the transgenic gene. Such regulatory sequences are, for example, promoters or terminators. Many functional promoters and terminators suitable for plants are known to those skilled in the art.

[0379] The present invention also includes vacuoles of cells according to the invention, and contents stored therein.

[0380] Furthermore, the present invention also relates to cell extracts derived from cells, preferably plant cells, particularly preferably sugar beet cells, and especially preferably from cells of one of the following crops: sugar beet, Swiss chard, or beet root. Plants cannot be regenerated from cell extracts.

[0381] This invention also covers plant genomes containing nucleic acids according to the invention.

[0382] Therefore, the sugar concentration of the cell extract can be increased relative to cells of the same species or crop but not according to the invention. This is particularly applicable to cases infected by *Cercospora*.

[0383] The present invention also covers the use of cell extracts for the production of sugar (sucrose) or for the production of juice (preferably beetroot juice).

[0384] The present invention also covers sugars, particularly sucrose, contained in cells and vacuoles according to the invention.

[0385] An additional aspect of the invention is a primary seed comprising seeds containing nucleic acids according to the invention. The nucleic acid molecules according to the invention can be present genetically or endogenously. The primary seed and seeds can be technically treated. Therefore, the invention also includes technically treated primary seeds and technically treated seeds. Various embodiments of technically treated primary seeds are explained in detail below, whereby the term primary seed also includes seeds: technically treated primary seeds can exist in a polished form. The outermost layer of the seed is thus removed, resulting in a more rounded shape. This is helpful in sowing, where the optimal uniform shape leads to a uniform distribution of the primary seed particles. Technically treated primary seeds also include granulated primary seeds. This embeds the primary seed within granular clumps to protect the primary seed contained therein and results in greater mass, making the granulated primary seeds more resistant to wind drift and therefore less susceptible to being blown away, while allowing for more precise positioning during sowing. In a preferred embodiment of the invention, all granulated primary seed particles in a designated batch or unit for sale have substantially the same shape and mass. A 5% deviation in diameter and mass is possible. However, the deviation is preferably no more than 1%. As a major component, granulated lumps may contain, for example, mineral compounds such as clay, bentonite, kaolin, humus, and / or peat. Binder materials such as polyacrylamide may be added. Additional possible components are referenced in US 4,067,141. Furthermore, granulated lumps may contain additional chemical agents that have a positive impact on cultivation in practice. These may be substances included in fertilizers. These include compounds rich in one or more of the following elements: nitrogen, phosphorus, and potassium (macronutrients). Therefore, fertilizer components may contain, for example, nitrate nitrogen, ammonium nitrogen, magnesium nitrate, calcium ammonium nitrate, monoammonium phosphate, monopotassium phosphate, and potassium nitrate. In addition, granulated lumps may contain fungicides, insecticides, and / or antifeedants. Fungicides may be thiram and / or hymexazol and / or other fungicides. Insecticides may be neonicotinoids. The neonicotinoids are preferably imidacloprid (ATC code: QP53AX17) and / or thiamethoxam (CAS No. 210880-92-5). In addition, the insecticide may also be deltamethrin (CAS No. 68359-37-5), lambda-cyhalothrin, or cypermethrin. It is worth mentioning that compounds included in seed dressings or granulated pellets are absorbed by the plant and exhibit systemic effects, thereby providing appropriate protection for the entire plant. Therefore, plants produced from granulated seeds including one or more insecticides differ from naturally occurring plants and exhibit better performance under biotic stress. In this context, the invention also covers mixtures of granulated pellets and seeds according to the invention. Furthermore, the invention also covers a method for producing granulated seeds according to the invention, the method comprising the following steps:

[0386] a) Provide plant seeds containing nucleic acids according to the invention,

[0387] b) Encapsulating plant seeds in granular aggregates, and

[0388] c) Allowing the granular agglomerates to dry or causing the granular agglomerates to dry, wherein the plant seeds may optionally be initiating or pre-germinating plant seeds, or the plant seeds may be allowed to initiate during step b). Optionally, the method may be carried out by an additional step d).

[0389] d) Package the embedded plant seeds obtained from step c) into a package. Examples of suitable packages are given elsewhere herein. Plant seeds may be, for example, seeds of beet plants (including sugar beets or red beets) as described herein.

[0390] Seed pellets may also contain beneficial bacteria. Preferably, the bacteria are alive and capable of proliferation. Symbiotic bacteria can have various positive effects on seed germination, including increased vigor, increased germination rate, and better resistance to stress. According to a preferred embodiment, microorganisms of the genus *Pseudomonas* spp. are introduced into the seed pellets. Additional strains of different bacteria may be added. Possible methods for improving microbial viability include the following steps:

[0391] a) Suspend one or more precultures of microbial cells in a polymer solution.

[0392] b) The microbial cells are immobilized by adding the solution from step a) dropwise into a multivalent ion solution.

[0393] This yields polymer particles.

[0394] c) Incubate the microorganisms encapsulated in the polymer particles in a liquid culture medium for at least 12 hours, preferably at least 24 hours, until a cell density increase of at least 2 to 10 log is obtained.

[0395] d) Collect the polymer particles, and e) Dry the polymer particles.

[0396] Granulated seeds containing the resistance gene according to the invention and also containing microorganisms obtained by the methods disclosed in this paragraph are part of the invention. Furthermore, the microorganisms may be encapsulated and / or embedded in an extracellular matrix containing natriuretic acid.

[0397] In this context, technically treated primary seeds also encompass seed-coated primary seeds. However, the present invention can be applied to seed-coated primary seeds in any form. Therefore, dry seed coating, wet seed coating, and suspension seed coating are also covered. Thus, the seed coating agent may also contain at least one dye (staining) to enable rapid differentiation of seed-coated primary seeds from uncoated primary seeds, and further ensure good visibility in the environment after sowing. The seed coating agent may also contain those agrochemicals described in the context of granular clumps. The present invention therefore includes seed-coated primary seeds in which the seed coating agent contains at least one anti-feeding agent, such as an insecticide and / or at least one fungicide. Optionally, so-called electric seed coating (seed coating by applying electrical energy) can be applied. Electric seed coating is not seed coating in the strict sense, but is very suitable for destroying plant pathogens adhering to seeds or primary seeds before planting. It is also advantageous that, in cases where more seeds or primary seeds are obtained than needed for cultivating the field, seeds or primary seeds treated solely by electric seed coating (without the use of agrochemicals) can be fed to animals.

[0398] An additional form of technically treated seed stock is coated seed stock. In this context, so-called coated and coated seed stock have also been proposed. The difference from granulated seed stock is that the seeds retain their original shape, a method particularly economical. This method is described, for example, in EP 0 334 258 A1. An additional form of technically treated seed stock is germinated or induced seed stock. Germinated seed stock is pretreated by germination, while induced seed stock has already been pretreated by induced (“germination”). The advantage of germination and induced seed stock is a shorter emergence time. Simultaneously, the emergence time after sowing is more strongly synchronized. This allows for better agro-technical processing during cultivation and especially during harvest, and additionally increases yield. In germination, the seed stock germinates until the radicle leaves the seed coat, after which the process is stopped. In induced seed stock, the process is stopped before the radicle leaves the seed coat. Compared to pre-germinated primary seeds, initiated primary seeds are less sensitive to the stress of re-drying and have a longer shelf life after such re-drying compared to pre-germinated primary seeds, which are generally not recommended for re-drying. In this context, technically pre-treated primary seeds also include those that have undergone initiation and re-drying. The germination process is described in US 4,905,411 A. Various embodiments of initiation are described in EP 0 686 340 A1. Furthermore, primary seeds can be granulated and initiated simultaneously in one process. This method is described in EP 2 002 702 B1. Additionally, granulated initiated primary seeds are also covered by this invention.

[0399] Technically treated primary seeds can additionally provide one or more of the explained herbicide resistances. This allows for further improvements in agricultural technology breeding, as technically treated primary seeds can be deployed in fields that have previously been treated with herbicides and are therefore weed-free.

[0400] In addition, the present invention also covers mixtures containing the original seed or seeds according to the present invention and the seed dressing clumps as defined above. Thus, the seed dressing clumps are preferably implemented as granular clumps as defined above.

[0401] For the storage of the original seed according to the invention, storage conditions that do not negatively affect the stability or shelf life of the original seed are preferably selected. Fluctuations in humidity can be particularly detrimental here. Part of the invention is a method for storing the original seed in a bag or container that is both waterproof and breathable. Such a bag or container may be designed as a carton or package. Such a carton or package may optionally have an internal moisture barrier. If the carton or package is designed as a double-layered carton, its stability is increased. Containers, bags, cartons, or packages containing the original seed according to the invention or the technically treated original seed according to the invention are also part of the invention. Similarly, storing the original seed according to the invention or the technically treated original seed according to the invention in such bags, containers, boxes, packages, or cartons is also part of the invention.

[0402] This invention also covers varieties containing the resistance gene according to the invention. Furthermore, it includes plants, seeds, and primary seeds of such varieties. Seeds and primary seeds of such varieties can be subjected to the techniques described herein (e.g., granulation). Suitable sugar beet varieties for introducing the resistance gene are, for example, BTS 7300N, BTS 2045, BTS 3750, DAPHNA, KORTESSA KWS, or SABATINA KWS. Named sugar beet plants are also examples of hybrid sugar beet plants. Suitable red beet varieties for introducing the resistance gene are, for example, Jolie, Scarlett (PV-9503), or Diaz, wherein Jolie and Diaz are also examples of hybrid red beet plants. Suitable Swiss chard varieties for introducing the resistance gene are, for example, Fluence, Ion, or Tesla / PV-9022. Suitable spinach varieties for introducing the resistance gene are, for example, PV-9210, PV-1194, or La Paz / PV-1237. Hybrid plants utilize the heterosis effect.

[0403] In one embodiment, the plant according to the invention is a hybrid plant or a double haploid plant. Hybrid plants and double haploid plants do not exist in nature and cannot be isolated from nature. In another embodiment of the plant according to the invention, the nucleic acid molecule according to the invention exists in a heterozygous or homozygous form. In the case of hybrid plants, the nucleic acid molecule may also exist in a hemizygous form. The invention also covers hybrid seeds and double haploid seeds containing the nucleic acid molecule according to the invention or the polypeptide according to the invention.

[0404] Another embodiment of the invention comprises a plant, preferably a sugar beet species, characterized by further increased resistance to *Cercospora*. This can be achieved, for example, by means of "gene stacking," i.e., using a dose-response effect to increase resistance. For this purpose, a plant according to the invention containing an allele conferring resistance to *Cercospora* is overconverted with that resistance allele to increase the amount of gene transcription in the plant. Alternative methods include gene editing / site-directed mutagenesis or tilling-mediated modification of the natural promoter of the resistance-conferring allele to increase its expression rate, or modification of the allele itself of the resistance-conferring LRR gene to increase its activity or stability. For example, such a method of increasing activity by modifying the resistance gene is described in WO 2006 / 128444 A2 and can be performed using techniques known to those skilled in the art. Additional methods may include fusing the nucleic acid molecule according to the invention with a heterologous promoter that exhibits higher activity than the natural promoter (especially after infection with *Cercospora*).

[0405] Additional embodiments of the invention relate to granulated seeds of sugar beet plants or portions thereof, or such plants, which are harvestable before bolting because no sugar beet plants bolt during the first 10, 11, 12, 13, 14, or 15 months, during which the development of the beet body is completed. Suitable varieties of sugar beet plants according to this paragraph, which generate resistance according to the invention, are, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0406] In one embodiment of the invention, the sugar beet plant or a portion thereof, or the granulated seeds of such plants, have a genome that allows the beet body to develop to at least 50%, 60%, 70%, 80%, or even 90% of its total mass as a fully developed plant. Suitable varieties of sugar beet plants according to this paragraph, which generate resistance according to the invention, are, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0407] In another embodiment of the invention, the sugar beet plant or a portion thereof, or the granulated seeds of such a plant, possess a genome that allows the beet body to develop via photosynthesis to a minimum mass of 200g, 250g, 300g, 350g, 400g, 450g, or 500g, and a maximum mass of 1000g, 1100g, 1200g, 1300g, 1400g, 1500g, 1600g, 1700g, 1800g, 1900g, or even 2000g. Suitable varieties of sugar beet plants according to this paragraph, which produce the resistance according to the invention, are, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0408] Additional embodiments of the invention relate to granulated seeds of sugar beet plants or portions thereof, or such plants, wherein the genome of the sugar beet plant allows the beet body to develop with a sucrose concentration of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or even 20%. Suitable varieties of sugar beet plants according to this paragraph, which generate resistance according to the invention, are, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0409] In one embodiment of the invention, the granulated seeds of sugar beet plants or a portion thereof or such plants include at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty or even at least thirty mutations relative to SEQ ID No. 1, 2 or 4.

[0410] A method for generating an organism comprising a mutant form of a nucleic acid molecule according to the above-given embodiment [1] and / or a mutant form containing a promoter of a nucleic acid sequence selected from: (a) SEQ ID NO:7, (b) a nucleotide sequence hybridized under stringent conditions to a sequence complementary to the sequence according to (a), and (c) a nucleotide sequence at least 70% identical to the sequence according to SEQ ID NO:7, wherein the method comprises the following steps:

[0411] (I) Providing an organism or cell containing nucleic acid molecules and / or promoters

[0412] (II) Increase the mutation rate of an organism or cell, or

[0413] Mutagenic organisms or cells

[0414] (III) Select an organism's phenotype that, as a result of mutation, exhibits altered resistance or altered resistance levels to *Cercospora beetroot*, or select an organism or cell genotype containing a mutation in a nucleic acid molecule and / or promoter, wherein the mutation is generated via step (II), and optionally...

[0415] (IV) Cell regeneration organism obtained via step (III).

[0416] The organism can be a plant. Preferably, the plant is a sugar beet. However, single-celled organisms such as bacteria can also be used. The bacteria can be *Escherichia coli* (E. coli). If the organism is a plant, the method can be applied both in vivo and in vitro. If the organism is a plant and the method is applied in vitro, a plant cell culture can be established, and an increase in mutation rate or mutagenesis can occur in the cell culture. An increase in mutation rate includes, for example, the application of mutagens, such as 5-bromouracil or ethyl methanesulfonate (EMS), or the application of physical mutagens, such as ionizing radiation or ultraviolet light. Mutagenesis also includes targeted mutagenesis. Targeted mutagenesis can be achieved through precise methods such as gene editing (explained further below). The regeneration of organisms from cells is explained in various standard references in cell biology. For example, plant regeneration is explained in the standard reference *Plant Biotechnology: Comprehensive Biotechnology*, Supplement 2 (Michael W. Fowler, Graham Warren, Murray Moo-Young-Pergamon Press, 1992). The regeneration of sugar from cell cultures using cultivated sugar beets is described in Lindsey & Gallois, *Transformation of Sugarbeet (Beta vulgaris), Agrobacterium tumefaciens*, *Journal of Experimental Botany*, 41.5 (1990): 529-536.

[0417] These references also describe how to establish plant cell cultures. As further explained above, the characteristics of the mutant forms of nucleic acid molecules and promoters are preferably attributed to the increased expression rate of the resistance-conferring nucleic acid molecules through mutation. This effect can also depend on the presence of several mutations. For example, two, three, four, five, or more mutations can be introduced into the promoter or nucleic acid molecule.

[0418] By introducing mutations, proteins that thus confer greater resistance or have a better effect can be constructed in cells. Thus, resistance can increase, for example, by at least 1%, 2%, 3%, 4%, 5%, or more, compared to control plants containing unaltered nucleic acids according to the invention. This increase can be measured as further explained below. Furthermore, resistance resulting from one or more mutations can increase at least one rating score. The determination of the rating score is explained elsewhere herein. Moreover, resistant proteins can confer altered effects due to mutations and, in some cases, can exhibit effects against such pathogens that have adapted to initial resistance mechanisms. In this context, the invention also covers variants of such mutations in the nucleic acids according to the invention and variants of mutations in the proteins according to the invention. Preferably, the invention covers variants that do not exist in nature and cannot be isolated from nature, ensuring that pathogens have no opportunity to adapt themselves to such variants. The methods described above for producing organisms containing mutant forms of nucleic acid molecules may further include the step of identifying those organisms or respective plants that have further increased resistance due to one or more mutations. If an increase in resistance has occurred, it can be determined by the rating score explained herein or by measuring the resistance level.

[0419] In addition to the methods described above for generating mutant forms of organisms containing nucleic acid molecules or promoters, the corresponding nucleic acids can also be chemically modified in their isolated state to achieve desired effects (such as those mentioned above). The advantage of this method is that it allows for even more precise editing of compounds. To this end, the following method is provided:

[0420] Generate chemically modified nucleic acid molecules according to the above-given embodiments [1] and / or promoters containing chemically modified nucleotide sequences selected from the following:

[0421] (a)SEQ ID NO:7;

[0422] (b) A nucleotide sequence that hybridizes with the nucleotide sequence according to (a) under stringent conditions;

[0423] (c) A nucleotide sequence that is at least 70% identical to the sequence according to SEQ ID NO:7;

[0424] This method includes the following steps:

[0425] (I) Provide the nucleic acid molecules as described above in isolated form.

[0426] (II) Chemically modify the nucleic acid molecule or promoter by one of the following steps:

[0427] (IIa) Mutagenesis

[0428] (IIb) Gene Editing

[0429] (IIc) Restriction and connection, insertion or deletion respectively.

[0430] Furthermore, in the case of allelic variants, chemical modifications can be generated by methods as described elsewhere herein. The gene editing given below step (II) above is equivalent to the term "genome editing." Optionally, the chemically modified nucleic acid molecule or the chemically modified promoter can then be introduced into the cell or stably integrated. With the aid of such cells, the chemically modified nucleic acid molecule and the modified promoter can be multiplied in the context of cell proliferation. They can then be isolated in large quantities, and expression analysis can be performed. Expression analysis is particularly suitable when the chemical modification involves the promoter. Cells can be harvested and the chemically modified resistance protein isolated for chemical analysis. If the cell containing the chemically modified nucleic acid molecule or the modified promoter is a plant cell, a complete plant can be regenerated from that cell. The methods described in this paragraph can be performed after the methods given above for generating modified forms of nucleic acid molecules and / or modified promoters, and the resulting variants are also part of the invention. Furthermore, plants containing chemically modified nucleic acid molecules or modified promoters are also part of the invention. Therefore, the invention also relates to plants obtained by this method. Furthermore, the invention also relates to chemically modified nucleic acid molecules and encoded polypeptides obtained by this method. These compounds are optimized forms of the original (unmodified) compounds, wherein the obtained resistance level (as further explained above) can be increased by at least 1%, 2%, 3%, 4%, 5%, or more percentage points, or by at least one rating score. In this respect, the methods for producing chemically modified nucleic acid molecules are also methods for optimizing nucleic acid molecules. The optimization methods may also include additional steps in which the modified variants of the nucleic acid molecule are identified, resulting in increased resistance in plants compared to the unmodified variant.

[0431] In another embodiment, the plant of the present invention additionally transgenically or endogenously contains a second nucleic acid molecule located at a different position in the genome that encodes a polypeptide capable of conferring resistance to *Cercospora* species expressing the polypeptide. For example, one or more resistance genes or resistance loci described in the prior art can be introduced into the plant of the present invention by means of hybridization, transformation, homologous directed repair, or homologous recombination in the plant, provided that they are not already present in the initial genotype. These include, for example, beet root rot resistance RZ1 (Lewellen, RT, IOSkoyen and AWErichsen, “Breeding sugarbeet for resistance to rhizomania: Evaluation of host-plant reactions and selection for and inheritance of resistance.” 50th Winter Congress of the International Institute for Sugar Beet Research, Brussels (Belgium), February 11-12, 1987, IIRB. Secretariat General, 1987), or beet root rot resistance RZ3 (WO 2014 / 202044), wherein the implementation schemes involving RZ3 are explained in more detail below paragraph

[94] .

[0432] The present invention further relates to a method for increasing the resistance of sugar beet species to the genus Cercospora, wherein the increase in resistance occurs in the absence of the resistance-conferring gene according to the present invention compared with plants of the same gene.

[0433] Increased resistance can be achieved by integrating the nucleic acid molecule according to the invention into the genome of at least one cell of a sugar beet species, and by regenerating the plant from that cell. Integration can be performed by sexual hybridization (e.g., with one of the aforementioned coastal beets) and subsequent selection, or by homologous directed repair or homologous recombination. The latter two methods are preferably supported by site-directed nucleases, which may be selected from, but are not limited to, CRISPR nucleases, including Cas9, CasX, CasY, or Cpf1 nucleases, TALE nucleases, zinc finger nucleases, broad-spectrum nucleases, Argonaut nucleases, restriction endonucleases (including FokI or variants thereof), recombinases, or two site-specific nicking endonucleases. Example 1 demonstrates the introduction of a resistance-conferring gene into red beet via CRISPR-mediated homologous recombination.

[0434] Furthermore, the present invention also covers a method for producing agronomic sugar beet plants of the genus *Saccharomyces* exhibiting improved resistance to *Cercospora beetula*, the method comprising infiltrating the plant with chromosomal regions conferring improved resistance to *Cercospora beetula*, wherein the chromosomal regions are mapped to positions between sequences represented by markers selected from s4p4293s01 and s4p4295s01 and sequences represented by markers selected from s4p4301s01 and sxh0678s01.

[0435] The feature is that the chromosomal region contains a nucleotide sequence encoding a polypeptide that confers resistance to *Cercospora betaine* in plants expressing the polypeptide, wherein the nucleotide sequence is selected from:

[0436] (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 3;

[0437] (b) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 2;

[0438] (c) A nucleotide sequence comprising a DNA sequence selected from SEQ ID No. 1 or SEQ ID No. 53;

[0439] (d) A nucleotide sequence that hybridizes under strict conditions with a nucleotide sequence complementary to the nucleotide sequence according to (a), (b), or (c);

[0440] (e) A nucleotide sequence that encodes a polypeptide that is different from the polypeptide encoded by the nucleotide sequence according to (a), (b) or (c) by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence;

[0441] (f) A nucleotide sequence encoding a polypeptide having at least 70% of the same amino acid sequence as the amino acid sequence according to SEQ ID No. 3;

[0442] (g) A nucleotide sequence that is at least 70% identical to the DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2;

[0443] Alternative methods include enhancing the expression of the nucleic acid molecules according to the invention in plants. This can be done by modifying natural promoters, wherein the modification is preferably carried out by gene editing or site-directed mutagenesis (mediated by site-directed nucleases) and optionally by repair models. Examples of such nucleases have already been cited above. Enhancement of the expression of the nucleic acid molecules according to the invention can also be achieved by fusing the nucleic acid molecules with a heterologous promoter that exhibits higher activity than the natural promoter, particularly after infection with *Cercospora*. Fusion can also be carried out via site-directed nucleases and repair models, and can be performed by direct insertion following a double-strand break.

[0444] As mentioned above, methods for increasing Cercospora resistance by modifying the nucleotide sequence of the nucleic acid molecule according to the invention can also lead to an increase in the activity and / or stability of the polypeptide according to the invention. For example, such a method of increasing activity by modifying a resistance gene is described in WO 2006 / 128444 A2 and can be performed using techniques known to those skilled in the art. This method will be explained in further detail below.

[0445] Alternatively, Cercospora resistance genotypes can be generated from Cercospora sensitivity genotypes by randomly or directed mutagenesis of the nucleic acid sequence of the sensitivity gene, thereby increasing Cercospora resistance. Figure 1 The paper presents examples of polymorphisms that distinguish between sensitive and resistant alleles.

[0446] For example, sensitive alleles can be modified by gene mutations using TALE nucleases (TALENs) or zinc finger nucleases (ZFNs), as well as CRISPR / Cas systems, which are described in particular by way of example in WO 2014 / 144155A1 (Engineering plant genomes using CRISPR / Cas systems) and Osakabe & Osakabe, Plant Cell Physiol., 56 (2015), 389-400. This can also be achieved using a method called TILLING (Targeted Induced Local Mutations in Genomes), which describes, for example, how to induce point mutations in sensitive genes and then select plants that exhibit suitable (i.e., resistant) mutations, such as barley resistant to yellow mosaic virus, as described in German patent application DE 10 2013 101 617; see DE 10 2013 101 617, pp. 4, 8, and 12, paragraphs

[0014] ,

[0026] , and

[0038] . The TILLING method is also described in detail in the publications of Henikoff et al. (Henikoff et al., Plant Physiol. 135, 2004, 630–636).

[0447] These methods preferably result in an improvement in resistance score by at least one rating score, and particularly preferably an improvement in resistance score by at least two, three, or higher. After mutagenesis of plant cells and subsequent regeneration of plants from the mutagenized plant cells, or after mutagenesis of the plant, plants exhibiting one or more mutations in endogenous nucleic acid molecules can be identified, such as… Figure 1As described. In this context, the plant mentioned according to the invention may be characterized by an increase in resistance by at least one rating score, preferably at least two or higher rating scores. Alternatively, compared to a control plant that does not contain the nucleic acid according to the invention, the plant according to the invention may have an increase in resistance, for example, at least 1%, 2%, 3%, 4%, 5% or more. The increase can be measured by inoculating a pathogen isolate separately on a healthy leaf and determining the infected surface after 15 days. A 5% reduction in the infected surface corresponds to a 5% increase in resistance. Additional parameters for making the measurement can be derived from the "Resistance Test" of the following embodiments.

[0448] An additional embodiment of the invention is a method for generating plants resistant to *Cercospora*, which can be carried out as follows: by transforming plant cells with a nucleic acid molecule, recombinant DNA molecule, or vector or expression cassette according to the invention, and by regenerating transgenic plants from the transformed plant cells (see Example 2), and by generating *Cercospora*-resistant genotypes through random or targeted mutagenesis of the nucleic acid sequence of a sensitivity gene as described above, or by hybridization and screening, for example using one of the aforementioned coastal sugar beets. The vector or expression cassette and the method for transforming plants have been described above.

[0449] As described above, the method for producing plants resistant to *Cercospora* can alternatively include: introducing a site-directed nuclease and a repair matrix into cells of a sugar beet species, wherein the site-directed nuclease is capable of generating at least one double-strand break in the cell's genome (preferably upstream and / or downstream of the target region), and the repair matrix contains nucleic acid molecules according to the invention. The method further includes culturing the cells under conditions allowing for homologous targeted repair or homologous recombination, wherein the nucleic acid molecules are introduced from the repair matrix into the plant's genome. Furthermore, plant regeneration from modified plant cells is also covered (see Example 1).

[0450] In a preferred embodiment, the target region is an allelic variant of the nucleic acid molecule according to the invention, wherein the allelic variant encodes a polypeptide that does not confer resistance to Cercospora. In another preferred embodiment, the allelic variant comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 6, and / or comprises a coding DNA sequence according to SEQ ID NO: 5 or a genomic DNA sequence according to SEQ ID No. 4.

[0451] As described in conjunction with the nucleic acid molecule according to the invention, substitutions, deletions, insertions, additions, and / or any other alterations may be introduced, which, individually or in combination, do indeed change the nucleotide sequence but perform the same function as the initial sequence (here, the nucleotide sequence of an allelic variant of the nucleic acid molecule according to the invention). Thus, in another embodiment, the invention includes a nucleotide sequence encoding a polypeptide representing a polypeptide derivative encoded by an allelic variant of the nucleic acid molecule according to the invention, or comprising an amino acid sequence of an allelic variant of the nucleic acid molecule according to the invention. A derived amino acid sequence having at least one substitution, deletion, insertion, or addition of one or more amino acids represents a derivative of the polypeptide, wherein the functionality of the encoded polypeptide / protein is preserved. Conventional methods known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, genome editing, etc., may be used to introduce nucleotide sequences through substitutions, deletions, insertions, additions, and / or any other changes (in alone or in combination with a gene), which do indeed change the nucleotide sequence but perform the same function as the initial sequence.

[0452] Regarding the amino acid sequence, after modification by the aforementioned methods, it also shares common structural domains and / or common functional activities. A nucleotide or amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide or amino acid sequence of a reference allele variant of a nucleic acid molecule according to the invention is defined herein as sufficiently similar. Therefore, the invention includes nucleotide sequences capable of hybridizing under stringent conditions with nucleotide sequences complementary to the nucleotide sequences of allele variants of a nucleic acid molecule according to the invention or nucleotide sequences encoding the corresponding amino acid sequences.

[0453] In another preferred embodiment, the method according to the invention is characterized in that the double-strand break occurs in an allele variant of the nucleic acid molecule according to embodiment [1], or at least one double-strand break occurs at a position of at least 10,000 base pairs upstream or downstream of the allele variant, wherein the allele variant encodes a polypeptide that does not confer resistance to Cercospora.

[0454] It will be apparent to those skilled in the art that many different sensitive sequences may arise, derived from the nucleic acid molecule according to the invention but not conferring resistance to *Cercospora*, such that the sequences listed above (SEQ ID No. 4, 5, and 6) should be considered merely examples of sequences, and the invention is not limited to the aforementioned allelic variants of the nucleic acid molecule according to the invention. Such allelic variants may comprise nucleotide sequences selected from:

[0455] (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to SEQ ID No. 6;

[0456] (b) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 5;

[0457] (c) A nucleotide sequence comprising the DNA sequence according to SEQ ID No. 4;

[0458] (d) Nucleotide sequences that hybridize under strict conditions with complementary sequences according to (a), (b), or (c);

[0459] (e) A nucleotide sequence that encodes a polypeptide that is different from the polypeptide encoded by the nucleotide sequence according to (a), (b) or (c) by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence;

[0460] (f) A nucleotide sequence encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as the amino acid sequence according to SEQ ID No. 6;

[0461] (g) A nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the DNA sequence according to SEQ ID No. 4 or SEQ ID No. 5;

[0462] As described above, quantitative inheritance via QTLs not only typically introduces desired resistance into plants but also often introduces undesirable traits (e.g., reduced yield) because additional genes unrelated to the positive trait of resistance are inherited. This increasingly occurs if, as in the case of Cercospora resistance, resistance is inherited via numerous resistance genes in previously available cultivars with minimal effects. Therefore, in a preferred embodiment, the introduction of the nucleic acid molecule according to the invention (which itself exhibits a dominant resistance effect) or a vector or expression cassette is independent of the introduction of undesirable traits, wherein yield is preferably not negatively affected. Furthermore, the invention covers plants obtained via such a method.

[0463] Although actual QTLs can be detected using QTL analysis previously known from existing techniques, the underlying genomic regions that reveal QTL effects also mediate the aforementioned drawbacks, which is why "linkage carryover" is also discussed in this paper. Furthermore, QTLs and their associated effects are not uniformly described in the corresponding existing techniques, and only weak effects are mediated, making the utilization of these results in breeding Cercospora resistant plants likely limited and largely uncertain. Now, by identifying the resistance genes described in this paper, targeted breeding and controlled integration of resistance genes into sugar beet gene banks can be achieved. This ensures the breeding and generation of novel Cercospora resistant cultivars that exhibit high resistance to the pathogen without negatively impacting sugar yield.

[0464] The present invention also relates to a method for identifying or detecting, and optionally selecting and / or providing, saccharide-grown plant species resistant to the pathogen *Cercospora*, characterized in that the method comprises the step of detecting the presence and / or expression of a nucleic acid molecule or polypeptide according to the invention in the plant or a sample / part thereof. The presence and / or expression of the nucleic acid molecule or polypeptide according to the invention can be tested using standard methods known to those skilled in the art (e.g., by means of PCR, RT-PCR, or Western blotting). Preferably, during PCR, the oligonucleotide or primer hybridizes with a genomic template comprising a nucleotide sequence as defined in step (i) of

[104] in such a manner / distance that the resulting amplification product contains up to 2000 bp, preferably up to 1500 bp, more preferably 1000 bp, and most preferably up to 500, 200, or 100 bp. The plants to be identified / tested, and optionally selected and / or provided, may be plants containing the resistance gene according to the invention and also containing the nucleotide sequence according to SEQ ID NO. 182 and / or the resistance allele according to the marker s4p8772s01, or may be plants obtainable from seeds deposited in NCIMB Aberdeen, UK with accession number NCIMB 43646. The methods and procedures according to this paragraph can also be used to exclude or disregard plants that do not carry the resistance gene according to the invention. This is particularly useful, for example, in breeding programs where some, but not all, progeny plants include resistance.

[0465] Furthermore, the identification method according to the invention also includes using molecular markers that detect one or more polymorphisms, by means of detecting at least one polymorphism between resistant and sensitive sequences (i.e., between the sequence of the nucleic acid molecule according to the invention and the sequence of an allelic variant of the nucleic acid molecule according to the invention). As described above, it will be apparent to those skilled in the art that there are many sensitive sequences, i.e., many sequences encoding allelic variants of the nucleic acid molecule according to the invention. One of these is detected by... Figure 1 The sequence comparison of the nucleotide sequence of the nucleic acid molecule according to the present invention is presented by way of example. Therefore, a preferred embodiment of the method according to the present invention includes the use of molecular markers for detecting polymorphism (especially diagnostic polymorphism) to detect... Figure 1 The invention describes or detects at least one polymorphism. The detection is preferably performed using at least one molecular marker for each polymorphism (particularly each diagnostic polymorphism). Those skilled in the art know which marker techniques to apply to detect the corresponding polymorphisms and how to construct molecular markers for this purpose (see Advances in Seed Science and Technology, Vol. I, Vanangamudi et al., 2008). Furthermore, this invention covers the description or detection based on… Figure 1 Polymorphic molecular markers, such as those used to detect based on Figure 1 Molecular markers for polymorphisms. Thus, markers that do not distinguish between various polymorphisms can also be used, as long as these markers can detect polymorphisms that occur in nucleic acid molecules according to the invention but are not present in sensitive allele variants.

[0466] Alternatively or additionally, the identification method according to the invention includes the step of detecting at least one marker locus in the nucleotide sequence of the nucleic acid molecule according to the invention or in its co-segregating region. Preferably, the co-segregating region is a genomic region in sugar beets that confers Cercospora resistance with the polypeptide according to the invention or that co-segregates with the nucleic acid molecule according to the invention; more preferably, the co-segregating region comprises and is flanked by markers sxh0678s01 and s4p0264s01, markers s4p4301s01 and s4p2271s01, markers s4p4301s01 and s4p4293s01, or markers s4p4301s01 and s4p4295s01. Thus, detection can be performed via method steps of binding at least one marker or at least one primer pair to the locus according to SEQ ID No. 74 or 75 (preferably to the locus according to SEQ ID No. 76 or 77), and optionally generating a signal thereon (e.g., a fluorescent signal or sequence amplification). Therefore, alternatively or additionally, the co-isolated region may contain sequences according to SEQ ID NO: 74 and / or 75, or SEQ ID NO: 76 and / or 77. Furthermore, the foregoing identification method also represents a method for selecting plants exhibiting resistance to the *Cercospora* species according to the invention. This selection method includes the final step of selecting resistant plants.

[0467] In this context, the present invention also includes developing or generating molecular markers suitable for detecting the aforementioned polymorphism between nucleic acid molecules (resistance alleles) and sensitivity allele variants according to the present invention, wherein the markers are preferably suitable for detection. Figure 1 The polymorphisms exhibited, or the construction of hybridization probes that specifically bind to the nucleotide sequence of the nucleic acid molecule according to the invention, or the generation of a pair of nucleic acid molecules suitable for amplification in PCR (regions specific to the nucleic acid molecule according to the invention) and thus for detection in plants or plant cells.

[0468] The present invention preferably includes a method for generating oligonucleotides of at least 15, 16, 17, 18, 19, or 20 nucleotides in length, preferably at least 21, 22, 23, 24, or 25, particularly preferably at least 30, 35, 40, 45, or 50, and especially preferably at least 100, 200, 300, 500, or 1,000 nucleotides, which specifically hybridize with the nucleotide sequence of a nucleic acid molecule or a complementary nucleic acid molecule according to the invention; or a pair of nucleic acid molecules (preferably in oligonucleotide form) suitable as forward and reverse primers to attach to regions specific to a nucleic acid molecule according to the invention, and for amplification in polymerase chain reaction (PCR), or suitable as forward and reverse primers to hybridize to regions in the sugar beet genome that are conferred Cercospora resistance by the polypeptide according to the invention or co-segregated with a nucleic acid molecule according to the invention. SEQ ID NO 98 and SEQ ID NO 99 provide examples of suitable primers for detecting resistance-mediated nucleotide sequences according to the invention. These two sequences construct a primer pair that can be used for PCR. The present invention also includes kits containing oligonucleotides or molecular markers according to the present invention.

[0469] The method for generating oligonucleotides initially comprises: comparing the nucleotide sequence of a nucleic acid molecule according to the invention with the nucleotide sequence of a corresponding nucleic acid molecule that does not confer resistance or a sensitive allele variant (which preferably has a nucleotide sequence according to SEQ ID No. 4 or 5); identifying sequence differences between the two nucleotide sequences; and generating a nucleic acid molecule (meaning oligonucleotide) that specifically binds to the nucleic acid molecule according to the invention but does not bind to a nucleic acid molecule that does not mediate resistance.

[0470] Furthermore, the oligonucleotides according to the invention can be linked to fluorescent dyes to generate fluorescent signals, for example, upon excitation by light of a corresponding wavelength. The fluorescent dye can be a fluorescent pigment. The oligonucleotides according to the invention can be coupled to other compounds suitable for signal generation. Such oligonucleotides do not exist in nature and cannot be isolated from nature. To produce such labeled oligonucleotides, the following steps are performed: DNA can be bio-orthogonally labeled. For this purpose, DNA can be labeled in vivo or in vitro with nucleoside analogs, which can then be coupled to a fluorophore, for example, in each Staudinger reaction. Alternatively, DNA can be chemically provided with a fluorophore. The oligonucleotides can be labeled using fluorophores, for example, those used in qPCR, DNA sequencing, and in situ hybridization, via phosphoramide synthesis. Furthermore, DNA can be enzymatically generated during a polymerase chain reaction with fluorescent nucleotides, or labeled with ligases or terminal deoxynucleotidyl transferases. DNA can also be detected indirectly via biotinylation and luciferin. For coupling, luciferin, fluorescent lanthanides, gold nanoparticles, carbon nanotubes, or quantum dots are used as fluorophores. One of the most commonly used fluorescent substances is FAM (carboxyfluorescein). Therefore, this invention covers oligonucleotides, and particularly primers with FAM tags. The FAM is preferably present as 6-FAM; however, other FAM variants, such as 5-FAM, may also be used depending on the desired wavelengths of emission and excitation. Examples of additional fluorescent tags are AlexaFluor, ATTO, Dabcyl, HEX, Rox, TET, Texas Red, and Yakima Yellow. Depending on the application, oligonucleotides modified with base or sugar-phosphate backbones are available. These include amino-dT, azide-dT, 2-aminopurine, 5-Br-dC, 2'-deoxyinosine (INO), 3'-deoxy-A, C, G, 5-Met-dC, 5-OH-Met-dCN, 6-Met-dA, etc.

[0471] Furthermore, the present invention also relates to a labeled chip (“DNA chip,” “assay,” or microarray) containing at least one oligonucleotide according to the invention suitable for detection. This labeled chip is suitable for use in one or more detection methods according to the invention.

[0472] The present invention also includes methods for producing proteins according to the invention. These methods include providing or cultivating a cell culture containing SEQ ID No. 2, and subsequently expressing the protein encoded by SEQ ID No. 2.

[0473] Furthermore, the present invention also relates to plants or portions thereof resistant to *Cercospora*, identified by the methods described above and selected, if applicable. In particular, the present invention relates to plant populations comprising plants obtained according to one of the methods described above, and preferably resistant to *Cercospora* leaf spot or *Cercospora* infection, and characterized by the presence of nucleic acid molecules according to the present invention. The population preferably has at least 10 plants, preferably at least 50 plants, more preferably at least 100 plants, particularly preferably at least 500 plants, and especially preferably at least 1,000 plants in agricultural cultivation. The proportion of plants in the population that do not carry the nucleic acid molecules according to the present invention and / or are susceptible to *Cercospora* leaf spot is preferably less than 25%, preferably less than 20%, more preferably less than 15%, even more preferably 10%, and particularly preferably less than 5%, if present.

[0474] Using the fine mapping described above, the location of genes conferring resistance to Cercospora in the coastal sugar beet genome can be determined, and the gene itself and surrounding sequence regions can be identified. This, in turn, represents the basis for developing DNA hybridization probes or genetic markers in target regions, which can be used to detect Cercospora resistance-mediated genes or to distinguish them from genes that do not confer resistance.

[0475] DNA hybridization probes can be derived from sequences of genes conferring resistance to Cercospora and can be used to screen genomic and / or cDNA libraries of desired organisms. These probes can be used to amplify identified homologous genes via known polymerase chain reaction (PCR) processes and to examine whether the gene conferring resistance to Cercospora is endogenously present in the organism or has been successfully introduced as a heterologous genetic element.

[0476] Those skilled in the art can employ conventional hybridization, cloning, and sequencing methods, as illustrated in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. Those skilled in the art can also synthesize and use oligonucleotide primers to amplify the sequence of the gene conferring resistance to *Ceratophyllum*. For specific hybridization to be achieved, such probes should be specific and have a length of at least 15 nucleotides, preferably at least 20 nucleotides. Detailed guidelines on nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part 1, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays.” Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, edited by Ausubel et al., Greene Publishing and Wiley Interscience, New York (1995).

[0477] Therefore, nucleic acid molecules with a length of at least 15, 16, 17, 18, 19, or 20, preferably at least 21, 22, 23, 24, or 25, particularly preferably at least 30, 35, 40, 45, or 50, and especially preferably at least 100, 200, 300, 500, or 1,000 nucleotides are the subject of this invention, wherein the nucleic acid molecule specifically hybridizes with the aforementioned nucleotide sequence according to the invention containing the gene conferring resistance to Cercospora. This also explicitly covers the range of 15 to 35 nucleotides.

[0478] Therefore, the present invention also relates to markers as oligonucleotides (particularly primer oligonucleotides). These are nucleic acid molecules comprising at least 15 nucleotides in length that specifically hybridize with nucleotide sequences as defined above.

[0479] In particular, the present invention covers a pair of nucleic acid molecules, preferably oligonucleotides or in the form of a kit containing the oligonucleotide pair, which are suitable as forward and reverse primers for hybridization to regions specific to the nucleic acid molecules according to the invention, and for amplification of them in polymerase chain reaction (PCR), or are suitable as forward and reverse primers for hybridization to regions in sugar beets whose genomes exhibit resistance to Cercospora conferred by the polypeptide according to the invention or co-segregation with the nucleic acid molecules according to the invention.

[0480] The following advantages can also be achieved through the present invention in the breeding and development of new resistant plant lines in the genus *Saccharomyces*. Sequence information and identified polymorphisms—which allow for differentiation between resistance and susceptibility alleles of the disclosed genes (i.e., between alleles conferring resistance to *Cercospora* and those not conferring such resistance)—make it possible to directly develop markers in the genes described above and in upstream and downstream regions, representing a significant advantage for plant breeders—particularly for the development of optimized superior lines without “linkage baggage.” Furthermore, knowledge of the sequence structure can be used to identify, for example, additional resistance genes that are homologous or orthologous—particularly for *Cercospora*.

[0481] Therefore, the present invention also covers methods for identifying additional nucleic acid molecules encoding polypeptides or additional proteins that confer resistance to *Cercospora* in plants expressing the polypeptide. Thus, those skilled in the art can use databases, suitable search profiles, and computer programs to screen for homologous sequences or perform sequence comparisons. Furthermore, those skilled in the art can obtain additional DNA sequences encoding *Cercospora* resistance proteins themselves using conventional molecular biology techniques and use them within the scope of the present invention. For example, suitable hybridization probes can be derived from the sequences of nucleic acid molecules according to the present invention and can be used to screen genomic and / or cDNA libraries of desired organisms. Those skilled in the art can employ conventional hybridization, cloning, and sequencing methods herein, for example, as listed in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. Using known sequences, those skilled in the art can also synthesize and use oligonucleotide primers to amplify the sequences of nucleic acid molecules conferring resistance to *Cercospora*.

[0482] In one embodiment, the invention therefore also covers a method for identifying a nucleic acid molecule encoding a polypeptide capable of conferring resistance to *Cercospora* in sugar beet species expressing the polypeptide. The method thus includes comparing, in the genotype of a sugar beet species, an amino acid sequence of the polypeptide according to the invention (which confers resistance to *Cercospora* in red beet) with an amino acid sequence from a sequence database, or with a sequence of an allelic variant of the polypeptide according to the invention. Furthermore, the method according to the invention includes identifying an amino acid sequence or allelic variant that is at least 80% identical to the amino acid sequence of the polypeptide according to the invention; introducing a nucleic acid molecule encoding the identified amino acid sequence or allelic variant into a sugar beet species; expressing the nucleic acid molecule in the plant; and optionally, subsequently verifying resistance to *Cercospora*.

[0483] As previously mentioned, additional proteins or their encoding genes that confer resistance to Cercospora can be identified via classical bioinformatics methods (database searches and computer programs for screening homologous sequences), i.e., homologs, analogs and orthologs that are at least 70% identical, preferably at least 80%, particularly preferably at least 90%, especially preferably at least 95%, or even 98% identical to the amino acid sequence of the polypeptide encoded by the nucleic acid molecule according to the invention.

[0484] Therefore, the term homolog means that the related genes (from two different plant species) have essentially the same function and a common ancestor, and thus typically show significant similarity in their nucleic acids or encoded amino acid sequences. However, there are also many genes that are homologous to each other without producing protein sequences with meaningful paired alignment. Conversely, the term analog describes genes or proteins that (again) have the same or similar functions but are not derived from the same structure, i.e., do not share a common ancestor. In this case, significant similarity usually cannot be established in their nucleic acid or encoded amino acid sequences, or, in the best case, in specific functional domains.

[0485] In the context of genome sequencing, homologs are classified more finely for labeling purposes. This has led to the introduction of the terms orthologs and paralogs. Orthologs are genes linked by speciation events. Paralogs are genes traced back to duplication events.

[0486] In the context of this invention, if a gene can confer resistance to the genus *Cercospora* in a plant, it is essentially a homolog, analogue, or ortholog. To verify this, methods known to those skilled in the art as described above are used, for example, to amplify the identified homolog, analogue, or ortholog by means of PCR, to clone it in an expression vector, to introduce it into a target plant or plant cell, and to examine the resistance.

[0487] As described above, the use of alleles of resistance genes in cis- or transgenic methods disclosed herein opens up the possibility of new resistant species in the *Sacchariformis* genus that exhibit higher resistance through dose-response, or where resistance breakage can be avoided and resistance development optimized by stacking the disclosed gene with other resistance genes. Genes can also be modified using tiling or targeted engineering to optimize codon selection, thereby increasing expression or developing new or modified resistance alleles. According to a preferred embodiment, the codon-optimized sequence or modified resistance allele is not naturally occurring but artificially synthesized. SEQ ID No. 94 provides an example of a modified genomic sequence in which codons at positions 16-18 are modified, but the encoded amino acid sequence remains unchanged, and corresponds to SEQ ID No. 3. SEQ ID No. 95 provides an example of a modified cDNA sequence in which codons at positions 55-57 are modified, but the encoded amino acid sequence remains unchanged, and corresponds to SEQ ID No. 3. SEQ ID No. 94 and SEQ ID No. 95 are also examples of hybridization sequences. An example of a modified resistance-conferring allele is given according to the amino acid sequence of SEQ ID No. 96, wherein the amino acid valine has been substituted with the amino acid leucine at position 209. The amino acid sequence according to SEQ ID No. 96 is encoded by the modified cDNA according to SEQ ID No. 97. These sequences are not naturally occurring but are artificially synthesized. When substituting amino acids in, for example, the resistance-mediating sequence according to SEQ ID No. 3, it is recommended to exchange amino acids within the following group:

[0488] a) Glycine, alanine, valine, leucine, isoleucine

[0489] b) Serine, cysteine, selenocysteine, threonine, methionine

[0490] c) Phenylalanine, tyrosine, tryptophan

[0491] d) Histidine, Lysine, Arginine

[0492] e) Aspartic acid, glutamic acid, asparagine, glutamine.

[0493] This invention also relates to the use in plants of the allele of the identified gene conferring resistance to *Cercospora*, which is genetically or molecularly superimposed with other genetic elements that may confer agronomically advantageous traits. The economic value of the cultivated plant can thus be significantly increased, for example, by increased yield compared to plants with the same genetics but not providing the nucleic acid molecule according to the invention. Furthermore, new crop areas for the plant may be opened up due to biological factors such as strong pathogen pressure, areas previously unavailable for cultivating this plant. Specifically, this invention relates to the use of the allele of the identified gene conferring resistance to *Cercospora* in methods for controlling infection by the pathogen *Cercospora betaine* in the agricultural or horticultural cultivation of *Betula* plants, for example, covering the identification and selection of *Betula* plants and / or the cultivation of such selected plants or their progeny by means of one of the aforementioned methods. Therefore, the present invention includes a method for cultivating sugar beet species, the method comprising, in a first step, providing sugar beet species resistant to *Cercospora* according to the invention, or producing sugar beet species by means of a production method according to the invention, or identifying and selecting sugar beet species by means of the aforementioned identification method according to the invention; and comprising, in a second step, cultivating plants from the first step, or deploying primary seeds of plants from the first step, or raising plants from the first step. The cultivation method thereby resists infection of the cultivated plants by *Cercospora*. The cultivation method may be part of a method for producing sugar. A method for producing sugar includes the steps of the cultivation method, and additionally harvesting the cultivated plants as a penultimate step, and extracting sugar from the aforementioned plants as a final step. The methods for identifying or detecting markers and oligonucleotides as described herein may be used, for example, for seeds deposited in NCIMB at Aberdeen, UK with accession number NCIMB 43646, or may be used for plants produced from deposited seeds. Furthermore, they can be used on a) plants as offspring of preserved seeds, or b) plants in which the resistance gene according to the invention has been transferred by one of the methods described herein (e.g., transformation, etc.), or c) to distinguish between plants containing the resistance gene according to the invention and plants lacking the resistance gene according to the invention.

[0494] The cultivation method may be part of a method for producing a primary species. A method for producing a primary species includes the steps of the cultivation method, plus vernalizing the cultivated plant as a penultimate step, and extracting seeds from the aforementioned plant as a final step.

[0495] The extracted seeds may optionally be granulated to obtain granulated primary seeds of sugar beet species. In this case, this is the method used to produce granulated primary seeds.

[0496] Furthermore, the method for producing primary species can be designed as a method for producing resistant primary species of *Cercospora*. The method for producing resistant primary species of *Cercospora* includes the steps of the method for producing primary species described above, and additionally, according to the method described herein, verification of the nucleic acid according to the invention as a final step in at least one, preferably at least 0.1% or at least 1% of the extracted seeds. Verification is particularly preferably carried out such that the seeds remain germinating. This means that extracting the DNA required for verification from the seeds does not neutralize the germination ability of the seeds. In this case, verification of the nucleic acid according to the invention can be carried out in a particularly large proportion of all extracted seeds. For example, verification can be carried out in at least 2%, preferably at least 3%, particularly preferably at least 4% of all extracted seeds.

[0497] Plants, their cells, or seeds or primary species according to the invention may additionally possess, or provide, agronomically advantageous characteristics. One example is tolerance or resistance to herbicides such as glyphosate, glufosinate, or ALS inhibitors. Tolerance to glyphosate or ALS inhibitor herbicides is preferred. Specific embodiments of glyphosate resistance are disclosed in US 7,335,816B2. Such glyphosate resistance can be obtained, for example, from primary species stored in NCIMB, Aberdeen (Scotland, UK) under accession numbers NCIMB 41158 or NCIMB 41159. Such seeds can be used to obtain glyphosate-resistant sugar beet plants. Glyphosate resistance can also be introduced into other species of the genus *Saccharum* via hybridization.

[0498] In the context of glyphosate resistance, the present invention therefore also covers plants, their cells, or seeds or original species, characterized in that they contain nucleic acids according to the invention, and further characterized in that:

[0499] a) A DNA fragment of genomic DNA of a plant, its parts, or its seeds can be amplified via polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID No. 81 and a second primer having the nucleotide sequence of SEQ ID No. 82, wherein the DNA fragment is at least 95%, preferably 100%, identical to the nucleotide sequence of SEQ ID No. 83, and / or

[0500] b) A DNA fragment of genomic DNA of a plant, its parts, or its seeds may be amplified via polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID No. 84 and a second primer having the nucleotide sequence of SEQ ID No. 85, wherein the DNA fragment is at least 95% identical, preferably 100% identical, to the nucleotide sequence of SEQ ID No. 86, and / or

[0501] c) A DNA fragment of the genomic DNA of a plant, its parts, or its seeds may be amplified by polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID No. 87 and a second primer having the nucleotide sequence of SEQ ID No. 88, wherein the DNA fragment is at least 95% identical, preferably 100% identical, to the nucleotide sequence of SEQ ID No. 89.

[0502] In some embodiments, the wild-type sugar beet epsp synthase has an amino acid sequence as provided in NCBI reference sequence XP_010692222.1. This wild-type epsp synthase can be mutated or modified (e.g., by one of the mutation or modification methods disclosed herein) to confer resistance to glyphosate. Such mutations can produce an endogenous allele encoding an epsp synthase having an amino acid different from proline at position 179. Preferably, the amino acid different from proline is serine. This invention covers plants containing mutated epsp synthases and resistance genes according to the invention. This includes plants containing the resistance gene of the invention and a nucleic acid molecule encoding an epsp synthase having an amino acid different from proline at position 179 and comprising an amino acid sequence selected from:

[0503] i) Sequence of SEQ ID NO:223

[0504] ii) Having an amino acid sequence at position 179 that is different from serine and proline, or

[0505] iii) A sequence that has at least 90%, more preferably 95%, and most preferably 99% identity with the sequence of i) or ii) over its entire length.

[0506] In this context, the mutant EPSP synthase can be encoded, for example, by the nucleic acid sequence according to SEQ ID No. 224. Further technical details regarding the use of the mutant EPSP synthase are available from WO2020064687(A1). Plants containing the resistance gene according to the invention and expressing the mutant EPSP synthase may also contain resistance to one or more ALS inhibitor herbicides as described herein. Plants containing the resistance gene according to the invention and expressing the mutant EPSP synthase may be non-transgenic plants.

[0507] In the context of this invention, the mutant EPSP synthase is an artificial compound that does not exist in nature. According to one specific embodiment, the plant containing the mutant EPSP synthase and the resistance gene according to the invention is not a product of a substantially biological process.

[0508] Specific embodiments of ALS inhibitor herbicide resistance are disclosed in document WO2012 / 049268A1. For example, such ALS inhibitor herbicide resistance can be obtained from the NCIMB deposit in Aberdeen, UK, accession number NCIMB41705. Furthermore, such ALS inhibitor resistance can be generated via tiling or site-directed mutagenesis (e.g., via gene editing), such as by using CRISPR / Cas, CRISPR / Cpf1, TALENS, or zinc finger nucleases. Therefore, the invention also covers plants, their cells, or seeds or species, characterized by containing nucleic acids according to the invention, and further characterized by mutations in the endogenous acetolactate synthase gene, which encodes an acetolactate synthase protein having an amino acid different from tryptophan due to a mutation at position 569. As a result of the mutation, the amino acid at position 569 is preferably alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, valine, or arginine. Position 569 is preferably defined via position 569 of SEQ ID No. 90. Furthermore, a specific sequence of the mutated acetolactate synthase gene in SEQ ID No. 91 is preferred. The mutated sequence of the acetolactate synthase gene, or the sequence according to SEQ ID No. 91, does not exist in nature and cannot be isolated from nature. Moreover, the mutation may exist in both heterozygous and homozygous forms in plants, their cells or seeds, or the original species. We recommend that the mutation exist in a homozygous form, as this can promote the development of a more stable or stronger resistance phenotype.

[0509] Those skilled in the art are familiar with many additional herbicides and their applicability from the prior art. They can invoke the prior art to understand which genetic elements will be used and in what manner to implement appropriate tolerance in plants.

[0510] Furthermore, herbicide tolerance has a synergistic effect, meaning that the use of herbicides can reduce weed occurrence. This is advantageous in combating Cercospora, as it is well known that the conidia (asexual spores) or pseudomatrix (mycelium) of Cercospora beetroot can survive on plant material for up to 2 years.

[0511] Another example of an agronomic advantage trait is additional pathogen resistance, where the pathogens can be, for example, insects, viruses, nematodes, bacteria, or fungi. For example, broad pathogen defense in plants can be achieved through different combinations of pathogen resistance / tolerance, as genetic elements can exhibit additive effects with each other. For example, many resistance genes used for this purpose are known to those skilled in the art as genetic elements. For example, US20160152999A1 discloses an RZ resistance gene against beet root rot caused by the fungal agent "beet necrosis yellow vein virus." Several resistances contained in a plant can have synergistic effects with each other. If a plant is first infected by a pathogen, its immune system is usually weakened, and the epidermis, which acts as an external barrier, is usually damaged, increasing the likelihood of further infection. An additional example of an agronomic advantage trait is cold hardiness or frost resistance. For example, plants exhibiting this trait can be sown earlier in the year or remain in the field longer, which can lead to increased yield. Here, those skilled in the art can also rely on existing techniques to find suitable genetic elements. Additional examples of agronomically advantageous traits include water use efficiency, nitrogen use efficiency, and yield. Genetic elements that can be used to confer such traits can be found in existing technologies.

[0512] Furthermore, many modifications for pathogen defense are known to those skilled in the art. In addition to the commonly described R gene family, the Avr / R method, Avr gene complementation (WO 2013 / 127379), R gene self-activation (WO2006 / 128444), or HIGS (host-induced gene silencing) method (e.g., WO2013 / 050024) may also be advantageously used. In particular, R gene self-activation may be important to this invention. For this purpose, a nucleic acid encoding a self-activated resistance protein that enables plants to develop pathogen resistance was created. This nucleic acid then has only a limited portion of the NBS-LRR resistance gene (such as the wb-R-gene), extending downstream from the 5' end of the NBS-LRR resistance gene coding region to the beginning of the NBS domain encoding the NBS-LRR resistance gene.

[0513] In this context, a method is also included that includes the step of removing a region of a nucleic acid according to the invention, the nucleic acid encoding an N-terminal region and starting from a p-loop in the NBS domain and extending to the end of the N-terminal region.

[0514] Resistance proteins encoded by such shortened nucleic acids are typically self-activating because they trigger an immune response in plants even in the absence of the relevant pathogen, thus increasing the plant's basal immunity. Furthermore, this invention encompasses such shortened nucleic acids and the polypeptides encoded therefrom.

[0515] Furthermore, the present invention also includes the use of alleles of the gene conferring resistance to Cercospora identified by the above method for combination with one of the aforementioned modifications or with the aforementioned genetic elements that can convey one or more agronomic advantages of a plant.

[0516] In addition to relating to the plants according to the invention, the invention also relates to seeds or progeny, or organs, plant parts, tissues, or cells thereof, in the production of products typically derived from sustainable raw materials (such as food and animal feed, preferably sugar or syrup (molasses), wherein molasses is also used in industrial applications, such as alcohol production or as a growth substrate for the production of biotechnology products), and in the production of materials or substances for use in the chemical industry (e.g., refined chemicals, pharmaceuticals or their precursors, diagnostic agents, cosmetics, bioethanol, or biogas). Examples of the use of sugar beets as a biofuel in biogas treatment plants are described in application DE 10 2012 022 178 A1; see, for example, paragraph 10.

[0517] The following examples illustrate the invention but do not limit its scope. Standard molecular biology methods were used unless otherwise stated; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Fritsch et al., Cold Spring Harbor Laboratory Press, 1989; Mayer et al., Immunochemical Methods in Cell and Molecular Biology (ed.), Academic Press, London, 1987; and Weir et al., Handbook of Experimental Immunology, Volumes I-IV, Blackwell (ed.), 1986.

[0518] The following will explain in detail some of the most important sequences according to the present invention:

[0519] -SEQ ID No. 1: Genomic DNA sequence of the gene conferring resistance to Cercospora from coastal sugar beet.

[0520] -SEQ ID No. 2: cDNA sequence of a gene that confers resistance to Cercospora, which does not exist in nature.

[0521] -SEQ ID No. 3: The amino acid sequence of a protein that confers resistance to Cercospora, as encoded by SEQ ID No. 1 or SEQ ID No. 2.

[0522] -SEQ ID No. 4: Genomic DNA sequence of a susceptible variant of the gene conferring resistance to Cercospora.

[0523] -SEQ ID No. 5: cDNA of a sensitive variant of the gene conferring resistance to Cercospora.

[0524] -SEQ ID No. 6: Amino acid sequence of a sensitive variant of the gene conferring resistance to Cercospora.

[0525] -SEQ ID No. 7: Natural promoter of the gene conferring resistance to Cercospora from coastal sugar beets.

[0526] -SEQ ID No. 8: Natural terminator of the gene conferring resistance to Cercospora from coastal sugar beets.

[0527] -SEQ ID No. 53: Sequence of a gene locus from coastal sugar beet containing the gene conferring resistance to Cercospora according to SEQ ID No. 1.

[0528] Example

[0529] Example 1: Introducing resistance-conferring genes into red beets via CRISPR-mediated homologous recombination

[0530] crRNA design and selection:

[0531] Using the CRISPR RGEN tool, suitable crRNAs for inducing Cpf1-mediated double-strand breaks were designed (Park J., Bae S., and Kim J.-S. Cas-Designer: A web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31, 4014-4016 (2015); Bae S., Park J., and Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014)). To this end, suitable prespacer sequences were sought within genomic DNA sequences of 500–1,300 bp in length, flanking the 5' and 3' ends of a Cercospora resistance gene from coastal sugar beets. To ensure the functionality of the endonuclease Cpf1 from *Lachnospiraceae bacterium* ND2006(Lb), a 24-nt prespacer sequence was selected, flanked by a basic prespacer adjacent motif (PAM) with the sequence 5'-TTTV-3' (V = G, C, or A). Suitable prespacer sequences were selected according to predetermined quality criteria for the tool and matched with potential off-target sites in the *Saccharomyces cerevisiae* reference genome. For further testing, only crRNAs with a maximum of 15 bases identical to the functional PAM, excluding the actual target sequence, were selected. Since the first 18 nt of the prespacer sequence is crucial for the detection and cleavage of the target sequence, unwanted cleavage within other genomic sequences can be excluded in this way (Tang, X., L. Glowder, T. Zhang, A. A. Malzahn, X. Zheng, D. D. Voytas, Z. Zhong, Y. Chen, Q. Ren, Q. Li, E. R. Kirkland, Y. Zhang and Y. Qi (2017), “A CRISPR-Cpf1 system for efficient genomeediting and transcriptional repression in plants.” Nat Plants 3:17018).In this way, four potential crRNAs (5'crRNA#1-4) can be identified in the 5'-flanking region of the resistance gene, and three crRNAs (3'crRNA#1-3) can be identified in the 3'-flanking region of the resistance gene (see Table A).

[0532] Table A: Selected target sequences within the 5'- and 3'-flanking DNA sequences of resistance genes in sugar beets. PAM is underlined.

[0533]

[0534] Cloning of genetic elements: To clone the cpf1 expression cassette and crRNA expression cassette, the detection sequence of the BbsI restriction enzyme that prevents cloning was first removed from the target vector pZFNnptII by introducing a point mutation (T to G). Mutagenesis was performed using a mutagenesis kit with two mutagenesis primers according to the manufacturer's instructions (see Table B).

[0535] Table B: Mutagenic primers used to introduce point mutations (T to G, indicated by underline) to remove the BbsI detection sequence.

[0536]

[0537] To express the Lbcpf1 gene in sugar beets, a codon-optimized DNA sequence for Arabidopsis thaliana was synthesized, containing a 5'-flanking PcUbi promoter sequence (SEQ ID No. 79) from parsley (Petroselinum crispum) and a 3'-flanking 3A terminator sequence from pea (Pea sp.). Restriction interfaces (HindIII) associated with cloning within the Lbcpf1 coding sequence (CDS) [SEQ ID No. 78] were removed by introducing a silent mutation (base exchange, without modifying the amino acid sequence) to avoid accidental cleavage within the coding region. Codon optimization was performed using the GeneArt algorithm from Invitrogene / ThermoScientific. To enable Cpf1 transport within the nucleus, the coding sequence for the nuclear localization signal (NLS) of SV40 was integrated into the cpf1 CDS at the 5' end, and the NLS of the nucleoplasmic protein was integrated at the 3' end. For the connection in the binary target carrier pZFNnptII ( Figure 2 The expression cassette has two HindIII-restricted interfaces on its flanks, which are then connected to pZFNnptII_LbCpf1. Successful insertion of the PcUbi::Cpf1::TPea expression cassette was verified by sequencing, in which the binding regions of the primers used for sequencing were located in the flanking vector region and within the expression cassette (see Table C).

[0538] Table C: Primers for sequencing the PcUbi::Cpf1::TPea expression cassette integrated into pZFNnptII

[0539] name Sequence 5'→3' pSeq_CRBM_F1 SEQ ID No. 25 pSeq_CRBM_R1 SEQ ID No. 26 pSeq_CRBM_F2 SEQ ID No. 27 pSeq_CRBM_R2 SEQ ID No. 28 pSeq_CRBM_F3 SEQ ID No. 29 pSeq_CRBM_R3 SEQ ID No. 30 pSeq_CRBM_F4 SEQ ID No. 31 pSeq_CRBM_R4 (SEQ ID No.32)

[0540] After transcription into plant cells, the crRNA should be cleaved by two flanking ribozymes. For this purpose, the precursor crRNA is flanked by the coding sequences of the hammerhead ribozyme and the HDV ribozyme (Tang, X., L. Glowder, T. Zhang, A. A. Amalzahn, X. Zheng, D. D. Voytas, Z. Zhong, Y. Chen, Q. Ren, Q. Li, E. R. Kirkland, Y. Zhang and Y. Qi (2017), “A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants.” Nat Plants 3:17018).

[0541] To achieve perfect ligation of the prespacer sequences at the coding sequence of the crRNA repeat, two BbsI detection sequences were integrated between the crRNA repeat and the HDV ribozyme, with the single-stranded overhangs used for cloning adjusted accordingly. To ensure identical expression intensity of cpf1 and crRNA, the crRNA ribozyme cassette was bound to the PcUbi promoter sequence at the 5' end and to a [one / the] 3A terminator sequence at the 3' end. For subsequent ligation in the target vector pZFNnptII_Cpf1, the crRNA expression cassette was flanked by two PstI interfaces and arranged as the synthetic DNA fragment. The prespacer sequences were synthesized as complementary oligonucleotides and annealed according to a standard protocol. The resulting 24-bp DNA fragment was flanked by ligation-associated 4-nt single-stranded overhangs (see Table D).

[0542] Table D: Sequences of oligonucleotides used to generate short 24-bp prespacer sequences. The 4-nt single-stranded overhangs used for ligation are the first four nucleotides of each sequence listed below.

[0543]

[0544]

[0545] The efficiency of four crRNAs was tested using Agrobacterium-mediated gene transfer in sugar beet leaves. The pZFNtDTnptII plasmid was co-transformed to examine transformation efficiency. Leaf explants were transformed via vacuum infiltration according to a standard protocol. Six days later, the fluorescence of tDT was examined using a fluorescence microscope, and leaf explants exhibiting heterogeneous fluorescence were discarded. Ten days after infiltration, the leaf explants were rapidly frozen in liquid nitrogen, homogenized, and genomic DNA was isolated using the CTAB method (Clarke, Joseph D., “Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation.” Cold Spring Harbor Protocols 2009.3(2009):pdb-prot5177). The efficiency of individual crRNAs was determined by an external service provider using NGS, comparing the frequency of insertion editing (e.g., insertion, deletion, or base exchange) with the frequency of unedited sequences in the genomic DNA.

[0546] As the synthetic DNA construct, the most efficient crRNAs (5'crRNA#3 and 3'crRNA#1) are arranged as a reverse expression cassette along with the aforementioned ribozyme, promoter, and terminator sequences. The entire DNA construct is flanked by two PstI restriction interfaces for cloning in the target vector pZFNnptII_LbCpf1. After crRNA insertion, the LbCpf1 and crRNA expression cassettes are ligated from the vector pZFNnptII_LbCpf1_crRNA to the pUbitDTnptII vector via HindIII.

[0547] As a repair template that should be integrated into the genome of sugar beet cultivation via homologous recombination, the resistance gene expression cassette has a 5'crRNA#3 binding sequence flanking the 5' end and a 3'crRNA#1 binding sequence flanking the 3' end. This allows for the excision of the resistance gene expression cassette from the plasmid via Cpfl. The entire DNA template was synthesized into a synthetic DNA fragment of 87,326 bp (SEQ ID No. 80) and directly used for transformation in the vector backbone. The resistance gene plasmid and the pUbitDTnptII_LbCpf1_crRNA plasmid were introduced into sugar beet callus cultures using gene cannon.

[0548] One day after transformation, transformation efficiency was determined using transient tDT fluorescence microscopy. Callus cultures were cultured on shoot induction medium without selection pressure (kanamycin-free), and the site-directed integration of the resistance-conferred cassette into regenerated shoots was examined. Genomic DNA was isolated using CTAB for this purpose. Integration of the resistance-conferred gene was amplified by PCR using primers pCRBM_F1 according to SEQ ID No. 47 and pCRBM_R1 according to SEQ ID No. 48 (see Table E), and the PCR products were subsequently sequenced using both primers. Shoots from which successful insertion of the expression cassette was verified were identified following analysis of the integration sites of the resistance gene. To verify the insertion of the desired target sequence in the genome, the flanking regions of the resistance gene expression cassette were amplified by PCR. Primer binding occurred within the resistance gene DNA sequence; binding of the second primer occurred outside the 5'- or 3'-flanking homologous regions of the inserted expression cassette (see Table E). The amplified DNA sequence was sequenced using the same primers, thus confirming integration at the desired location. To exclude binding of primers pCRBM_F1 (SEQ ID No. 47), pCRBM_R1 (SEQ ID No. 48), pCRBM_R2 (SEQ ID No. 50), and pCRBM_F3 (SEQ ID No. 51) in sequence-similar regions of the genome, all primer sequences were compared with the sugar beet genome beforehand. For primer pCRBM_F3 (SEQ ID No. 51), it was impossible to select a nucleotide sequence that would exclude binding to the wild-type sequence. Therefore, the 3' flanking region was amplified in shoots that tested positive for all resistance genes, and site-specific insertion was specifically verified by subsequent sequencing. The resulting PCR product differed from the wild-type sequence by 18 bp. To enable complete sequencing of the amplified sequence, the PCR product was additionally sequenced using third primers (pCRBM_S2, pCRBM_S3; see Table E) that have binding sites within the amplified sequence. To rule out non-specific binding of primers pCRBM_F1 (SEQ ID No. 47), pCRBM_R1 (SEQ ID No. 48), and pCRBM_R2 (SEQ ID No. 50) within the wild-type genome, the nucleotide sequences were compared with an internal reference genome of cultivated sugar beet. The binding of the primers to the genome sequence of wild-type cultivated sugar beet was further tested using PCR.

[0549] To prevent the integration of resistance genes into other regions of the genome, targeted amplification (TLA) is performed at the target site.

[0550] Table E: Primers used to verify the insertion of the resistance gene expression cassette into the desired integration site.

[0551]

[0552] In addition to verifying the resistance gene expression cassette and successfully inserting it into the sugar beet genome, unwanted integration of plasmid DNA was also examined. For this purpose, the presence of plasmid DNA in the genomic DNA (which verified successful insertion of the resistance gene at the desired target site) was examined by PCR. Subsequently, the sequence regions within cpf1, the crRNA ribozyme cassette, and the tDT were amplified using the primers listed in Table F, followed by sequencing.

[0553] Table F: Primers used to verify the plasmid-specific sequences stably integrated into the genome of sugar beet sprouts grown for regenerated sugars.

[0554] Example 2: Introducing resistance-conferring genes as transgenic plants into red sugar beets via gene transformation.

[0555] Transgenic methods for generating resistance to Cercospora are not only used to validate the LRR gene as a substitute for resistance-conferring genes, but also as a means to generate transgenic resistance events that confer novel Cercospora resistance or improve existing Cercospora resistance.

[0556] The binary vector pZFN-nptII-LRR was generated using the following standard cloning procedure: Within the T-DNA of this vector, the cDNA of the resistance gene according to SEQ ID NO 2 was cloned along with its natural promoter sequence. The T-DNA also included the neomycin phosphotransferase II (nptII) gene, which confers bandwidth resistance to aminoglycoside antibiotics such as kanamycin or paromomycin. This antibiotic resistance is used for selecting transgenic plant cells and tissues. The NOS promoter and pAG7 terminator are located flanking the nptII gene. The backbone of the binary vector also contains colE1 and pVS1 sources for plasmid replication in *Escherichia coli* or *Agrobacterium tumefaciens*. The aadA gene confers streptomycin / spectinomycin resistance for bacterial selection. The pZFN-nptII-LRR plasmid was transformed into *Agrobacterium* strain AGL-1 using a standard procedure.

[0557] The transformation of sugar beet was carried out according to Lindsey & Gallois (1990), “Transformation of sugar beet (Beta vulgaris), Agrobacterium tumefaciens.” Journal of Experimental Botany 41.5, 529-536. For this purpose, “micropropagated shoots” of genotype 04E05B1DH5 without carrying the resistance gene according to the invention were used as starting material. The shoots were propagated in the appropriate medium according to Lindsey & Gallois (1990). To induce as much meristematic tissue as possible, the “shoots” were transferred to different media (see Lindsey & Gallois (1990)) and incubated in the dark at approximately 30°C for several weeks. Agrobacterium strain AGL-1 carrying the vector pZFN-nptII-LRR was used. Figure 3 The explants were cultured in an additional medium containing the appropriate antibiotics for selection (see Lindsey & Gallois (1990)). Sections of meristems based on the buds to be treated were incubated with Agrobacterium in the additional medium for several hours (see Lindsey & Gallois (1990)). The plant explants and Agrobacterium were co-cultured in the medium in the dark for at least 2 days (see Lindsey & Gallois (1990)), and the inoculated explants were then incubated in the additional medium in the dark for about 2 weeks (Lindsey & Gallois (1990)). Subsequently, the explants were further propagated in the additional medium (see Lindsey & Gallois (1990)) and subcultured to allow selection of transgenic tissues. To conclude the selection phase and reduce the degree of chimerism, the green “buds” were transferred to medium H and propagated for 2 weeks. Leaf material was then extracted from the green, growing “buds” and the presence of transgenes was examined by means of PCT. Suitable buds were rooted in medium I and then transferred to a greenhouse for the production of T1 primary strains. Furthermore, leaf material derived from these buds was used to analyze the expression of transformed resistance genes.

[0558] Expression level analysis

[0559] RNA was isolated from the leaves of the in vitro "bud" and used in qRT-PCR. qRT-PCR was performed according to Weltmeier et al., 2011 (see Background Art). Measurements were normalized against the reference gene PLT3_075_F09 (see Weltmeier et al., 2011). Expression was determined using the following primer sequences:

[0560] sequence Size [No. Nucleotide] <![CDATA[T m [C°]]]> Amplification product size [No. nucleotides] SEQ ID No. 92 21 59,8 170 SEQ ID No. 93 21 58,9 170

[0561] Resistance testing of sugar beets inoculated with *Cercospora beta* under greenhouse conditions:

[0562] A pure culture of *Cercospora betta*, known to be highly toxic, was propagated on vegetable juice agar in petri dishes (9 cm diameter) under near-ultraviolet (NUV) light at 20°C. After 14 days, the surface of the mold-grown agar was submerged in 10 ml of sterile water in each petri dish, and conidia and mycelial fragments were carefully scraped off with the aid of the test medium. Plants were inoculated using an inoculum density of 20,000 conidia / mycelial fragments per ml, supplemented with 0.1% TWEEN 20. At the time of inoculation, the plants had been cultured under greenhouse conditions for 8 to 9 weeks. The apical and ventral sides of the leaves were treated with the inoculum. The plants were then cultured at 25°C, 18 h / 6 h light / dark, and approximately 100% humidity for 5 to 7 days. The first *Cercospora* symptoms appeared on the beet leaves after 12 to 14 hours. Symptoms were assessed regularly on individual plants with the aid of the grading assessment shown in Table 1A. The results are shown below.

[0563] Table G: Transgenic verification results of the function of the resistance gene according to the present invention in transformed plants;

[0564] LSD = minimum significant difference; dpi = days post-infection.

[0565]

[0566]

[0567] Transgenic verification results of the resistance gene according to the present invention (see Table G)

[0568] Test group 1 represents the negative control. The genotype is the same as test groups 2 to 11, but no transformation has occurred. Therefore, expression is undetectable. Test group 4 was transformed, but expression was undetectable. Test groups 2, 3, and 5 to 11 represent transformants carrying the resistance gene according to the invention solely due to transformation. Test group 12 represents breeding lines containing the non-transgenic form of the resistance gene according to the invention. The grading scores of all lines were determined after inoculating the plant material with *Cercospora beetroot* as described above. Test group 12 showed the highest resistance, with a final value indicating 5.19.

[0569] The transgenic lines show a grade rating according to the table below:

[0570] Table H: Grading of transgenic lines in Table G

[0571]

[0572] Table H shows only the grading scores for the transgenic validation groups. First, the average scores for all transgenic test groups (excluding group 4) are shown. The grading scores for those transgenic lines showing an expression level of at least 10 are given below (groups 3, 8, 9, and 10; see Table G). The final grading score here is 5.91. This is significantly higher than the resistance of the negative control group 1 (which only had a grading score of 6.46) (minimum significant difference = 0.4; see Table G). The best transgenic test group (group 8) showed even better resistance, as its grading score was 5.48 (see Table G).

[0573] It is worth mentioning that the expression level of the transgenic insert may be affected by the integration locus. When expression levels are measured in vitro, the actual expression levels under infection conditions may be higher, especially when the resistance gene is controlled by a pathogen-inducible promoter.

[0574] Statistical evaluation of transgenic validation results

[0575] Table I: Statistical Cluster Analysis

[0576] Test group Clustering 8dpi Clustering 11dpi Clustering at 13dpi Clustering at 15dpi 1 ab a a ab 2 e de bc cd 3 e ef c bcd 4 bc bcd bc bcd 5 cd abc a abc 6 a ab a ab 7 ab a a a 8 de ef c e 9 e de bc d 10 cd cd b d 11 ab a a a 12 de f d e

[0577] Table I shows the statistical evaluation of the rank scores contained in Table G. Each letter represents an assignment to a statistical group. For example, it is clear that after the final evaluation (15 dpi), test group 8 (transgenic validation) is in the same cluster as test group 12 (resistance source), but in a different cluster than test group 1 (negative control). Therefore, test group 8 is significantly different from test group 1, but not significantly different from test group 12.

[0578] In addition, box plot analysis was performed. From Figure 4-7 A box plot illustration can be obtained.

[0579] Example 3: Production of resistant sugar beet plants according to the present invention based on genetic material obtained from coastal sugar beets

[0580] The procedure described below is based on collecting wild sugar beet materials to generate a Cercospora resistance gene library. The following table lists the coastal sugar beets imported from Tibet that were used as starting materials for the breeding program.

[0581] Table 2: Coastal sugar beets deposited in the National Geological Center for the breeding program and their resistance ratings to *Cercospora*; the first four columns on the right show the accession number, where specific accessions may have different accession numbers depending on the conservation facility (USDA GRIN = US Department of Agriculture Germplasm Resources Information Network; IDBBNR = International Database for Beta; DEU001 = Plant Genetic Resource Collection; IPK = Leibnitz-Institut für Pflanzengenetik und Kulturpflanzenforschung).

[0582]

[0583]

[0584]

[0585] As is evident from Table 2, the obtained genetic material had previously shown inconsistent levels of resistance to *Cercospora*, and the degree of resistance varied across different studies. For example, the imported “PI 120704” scored 1 in one study and 9 in another. Since this publicly available data appeared unreliable, seed material obtained from the imported stock was cultivated, and *Cercospora* resistance was screened for phenotypic resistance in the resulting plants. Approximately 150 partially resistant plants were selected. However, since the resistance observed in each plant may be the result of a large number of genes, each contributing relatively little, the opportunity to identify individual genes suitable for establishing resistance or increasing resistance levels in a measurable manner is limited. It was decided to use a free-pollination scheme to cross approximately 150 resistant plants with each other. This method also allows for recombination within the genetic material. Crossover and selection were repeated for several generations to improve resistance levels. The best offspring were cloned and prepared for genetic mapping methods. The localization of the resistance described herein was combined with dense phenotypes. By establishing populations of over 4,000 dividing progeny groups and developing specialized recombination screening, the target region was narrowed and thus further segregated through analysis of informative recombinants (genotype and phenotype) in a series of resistance tests. This genetic mapping, along with the creation of a physical map using accompanying WHG sequencing (“whole genome sequencing”), comparative BAC (Bac-by-Bac) sequencing, and bioinformatics analysis, led to the identification of three recombinant genotypes of the resistance gene (one with one recombinant in adjacent genes, and the other with two recombinants in adjacent genes). Given the specific requirements, the inventors placed highly repetitive structures within the target region, containing tandem repeat sequences with very high sequence homology, which made marker development and therefore the identification of informative recombinants more difficult. The following steps are particularly crucial for locating the genetic structure of the resistance gene:

[0586] - Development of the tags s4p0264s01, s4p2271s01, sxh0678s01, s4p4293s01, s4p4295s01, and s4p4301s01 (see Table 1B).

[0587] - Fine-tuning is achieved by combining with dense phenotyping. In greenhouse testing, the phenotype is validated using 90-180 offspring per plant and dense statistical methods (e.g., t-tests, power analysis, etc.).

[0588] - Identify and sequence BAC clones from a BAC library of resistant genotypes.

[0589] - Sequence evaluation, and comparison of sequences and proteins between RR (i.e., resistance) and ss (i.e., sensitivity) genotypes; thus, due to sequence complexity, it is not always possible to perform an explicit assembly of RR and ss sequence data.

[0590] Within the framework of the breeding program, resistance derived from coastal sugar beets was crossed with superior sugar beet lines. Several backcrosses via marker-assisted selection allowed the transfer of the resistance gene into established sugar beet germplasm. Surprisingly, no undesirable effects on sugar yield, etc., were observed. Subsequently, a proof-of-concept for the resistance gene within sugar beets was established by transforming and generating transgenic sugar beets containing the resistance gene (see above). Following this successful proof-of-concept, the sugar beet germplasm containing the resistance gene can be used to generate Cercospora-resistant sugar beet varieties.

[0591] Example 4: Screening the initial entry into Tibet for the identified resistance genes

[0592] After identifying the resistance gene, marker screening was used to identify genetically sourced materials (according to Table 2) to identify those carrying the resistance gene. The number of analyzed plants for each entry depended on seed availability and is given in the table below.

[0593] Table 3. Number of each plant species introduced into Tibet analyzed for the presence of identified resistance genes.

[0594]

[0595]

[0596] Each of the given imported plants was screened using 572 SNP markers located at the 5' and 3' positions of the resistance gene. Due to the large number of markers, haplotype patterns were obtained. However, none of the imported plants used as starting material showed a haplotype of the line CRBM carrying the identified resistance gene. The most similar haplotype was found to be imported 48819 (DEU001 nomenclature) / 3555 (IDBBNR) (see Tables 2 and 3). The table below shows the extraction of the entire marker analysis, including the 5' and 3' positions of the resistance gene.

[0597] Table 4. Comparison of the resistant strains according to the present invention and 14 plants from SEQ ID NO 14 (del = deletion, ins = insertion, Pos start = start position of molecular marker on the genetic chain, * = position of the resistance gene according to SEQ ID NO 1) by SNP marker analysis

[0598]

[0599]

[0600] The results of marker analysis (as exemplified by the data given in Table 4) show that the resistance gene according to the invention cannot be traced back to one of the imported varieties according to Table 2. Even the imported variety 48819, which has the strongest marker overlap with the resistant line according to the invention, shows significant differences. Notably, deletions were detected within the resistant line, while imported variety 48819 showed deletions at the same location. This may indicate significant genetic recombination at this locus during the generation of the resistance gene according to the invention. This hypothesis also explains why the resistance gene cannot be traced back to the starting material of the breeding program.

[0601] Example 5: Production of Cercospora resistance protozoa

[0602] The resulting sugar beet germplasm containing the resistance gene (result of Example 3) can be used to generate Cercospora-resistant sugar beet varieties. For this purpose, the gene is transferred via hybridization with a DH parent line, which is then hybridized with a DH parent line from another hybridization breeding bank. The result is a hybrid variety containing resistance to Cercospora according to the invention. The seeds of this variety are separated from each other (isolated), cleaned, and polished. The seeds are then subjected to initiation and granulation as described in EP2002702A1. The resulting primary seeds are packaged in cardboard boxes containing a layer as a moisture barrier. The resulting primary seeds are suitable for sowing, growing, harvesting, and subsequent industrial sugar production.

[0603] Original table

[0604]

Claims

1. A method for identifying a Beta plant or a Spinacia plant that is resistant or tolerant to Cercospora, characterized in that The method comprises detecting at least one marker locus in a co-segregation region, wherein the co-segregation region is a genomic region co-segregating with the resistance to Aspergillus conferred by the polypeptide of the amino acid sequence of SEQ ID No. 3, or a genomic region co-segregating with the nucleotide sequence of SEQ ID No. 1, 2 or 53, and wherein the co-segregation region comprises the markers s4p1395s01 and s4p0421s01 and is flanked by the markers s4p1395s01 and s4p0421s01.

2. The method according to claim 1, characterized in that The method comprises the further step of selecting the Aspergillus resistant plant.

3. The method according to claim 1 or 2, wherein the detection is based on at least one polymorphism or single nucleotide polymorphism.

4. The method of claim 3, further characterized by a) the at least one polymorphism or single nucleotide polymorphism is genetically linked to the nucleotide sequence, b) the at least one polymorphism or single nucleotide polymorphism is located within 2562 kbp, 2300 kbp, 2100 kbp, 1900 kbp, 1700 kbp, 1500 kbp, 1300 kbp, 1100 kbp, 900 kbp, 700 kbp, 500 kbp, 300 kbp, 100 kbp, 50 kbp, 25 kbp, 10 kbp, 5 kbp or 1 kbp or less of the nucleotide sequence, c) the at least one polymorphism or single nucleotide polymorphism is detectable in seeds of the accession number NCIMB 43646 deposited at the NCIMB, Aberdeen, UK, or d) the at least one polymorphism or single nucleotide polymorphism is part of the co-segregation region, wherein the nucleotide sequence is the nucleotide sequence as defined in claim 1, and wherein the co-segregation region is the co-segregation region as defined in claim 1.

5. The method according to claim 1, further comprising the following steps ending with the detection: a) providing a plant, plant tissue, plant seed, or plant cell, and b) extracting DNA from the plant, plant tissue, plant seed, or plant cell.

6. The method according to claim 1, involving the use of at least two oligonucleotides.

7. The method according to claim 6, wherein the oligonucleotides are suitable for use as primers in a PCR and are capable of hybridizing to a genomic spacer region comprising the markers s4p1395s01 and s4p0421s01 and flanked by the markers s4p1395s01 and s4p0421s01.

8. The method according to claim 1, involving a PCR, wherein the PCR involves two allele specific forward primers, and wherein the detection step involves fluorescence resonance energy transfer, and wherein the presence, absence or kind of fluorescence is determined by a sensor.

9. The method according to claim 8, involving one universal reverse primer.

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