Plant resistance genes and means for identifying them

Abstract

The provision of the Circospora resistance mediating gene according to the present invention enables more efficient breeding or the development of new resistance lines against Circospora leaf spot disease. In particular, the dominant resistance effect in the target plant is caused solely by the characteristics of the identified gene. The Circospora resistance mediating gene and the above-described embodiments of the present invention provide further applications for the development of new resistant cultivars, for example, the use of alleles of the resistance gene in cis- or trans-genetic approaches.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid molecule encoding a polypeptide that can confer resistance to Circospora, particularly the fungus Circospora beticola, to plants expressing the polypeptide, especially plants of the Beta vulgaris species, and to a polypeptide encoded by the nucleic acid molecule according to the present invention. In particular, the nucleic acid molecule according to the present invention is characterized in that the resistance effect against Circospora conferred by the polypeptide is dominant. Furthermore, the present invention relates to Circospora-resistant plants, plant cells, plant organs, plant tissues, plant parts, or plant seeds or offspring that include the nucleic acid molecule or a part thereof as an endogenous gene, edited gene, or transgene. Furthermore, the present invention also includes a method for increasing resistance to Circospora in plants of the Beta vulgaris species, and a method for producing, identifying, and optionally selecting Circospora-resistant plants. The present invention also includes a method for monitoring invasion by the pathogen Cercospora beticola, as well as oligonucleotide probes and primers for hybridization with nucleic acid molecules according to the present invention.

[0002] Background of the Invention Cercospora leaf spot is one of the most important and widespread leaf diseases of various plants, including the Beta vulgaris and Spinacia oleracea species. It is caused by the fungus Cercospora beticola. Plants infected with this disease typically form small, relatively round leaf spots (2 - 3 mm) that are light gray in the center and surrounded by a reddish-brown margin. In severe attacks, the leaf spots overlap and the entire leaf blade withers. Small black dots (pseudothecia) are visible within fully formed spots, and under humid conditions, a gray, felt-like coating (conidiophore containing conidia) is formed mainly on the underside of the leaf. Severely infected leaves first turn yellow, then brown and die. New leaf growth occurs in parallel, but the leaves become diseased again and die. Initially, damage symptoms are only seen in individual plants, but as the disease spreads, the formation of persistent infection foci often occurs. Further spread across the field occurs by rain and wind. In the case of chard plants, damaged leaves are unacceptable to consumers, and even low disease pressure can lead to significant yield losses.

[0003] The pathogen Cercospora beticola was first reported in Italy in the late 19th century. Severe invasion, which can be induced by humid climates, early furrow closure, high likelihood of infection from the previous year, or heavy irrigation, can result in crop losses of up to 40%. These losses are attributed to reduced beet yields and decreased sugar content. 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 (MJC Asher, B. Holtschulte, MR Molard, F. Rosso, G. Steinruecken, R. Beckers, eds.). International Institute for Beet Research, Brussels, Belgium, 215 pp.). Intercropping or fungicides are often used to control this disease. Chemical control of Cercospora beticola with fungicides burdens farmers and pollutes the environment. Furthermore, the treatment of edible chard leaves with chemicals reduces consumer support. Repeated application of fungicides further increases the selective pressure on fungicide-resistant Cercospora beticola strains, which is contrary to sustainable agricultural practices. It is worth noting that in recent years, Cercospora beticola stems have emerged that exhibit resistance to one or more fungicides.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 issue has become so serious that the German Federal Office for Consumer Protection and Food Safety (BVL) has granted exemption from liability for copper-based fungicides to combat Cercospora. However, copper-based fungicides are generally considered harmful to humans and the environment (depending on the dose). Copper is a heavy metal that can accumulate in soil.

[0004] Indirect measures are taken by selecting cultivars with healthy leaves and cultivating beets in a crop rotation of at least three years. Significantly better control of invasiveness can be achieved by combining tolerant or resistant cultivars. Since 2000, less susceptible circospora-resistant beet cultivars have been available on the market (Steinruecken 1997, “Die Zuechtung von Cercospora-resistenten Zuckerrueben.” [“The breeding of Cercospora-resistant sugar beets.”], Vortraege fuer Pflanzenzuechtung [Lectures on Plant Breeding], Volume 37, Lecture symposium, March 4-5, 1997, Kiel). These cultivars possess quantitative resistance to circospora beticola. The resistance of these cultivars is inherited quantitatively based on several genes, but the exact number of genes involved in resistance is unknown. See Weiland and Koch (2004), Sugarbeet leaf spot disease (Cercospora beticola Sacc.), The Plant Journal, 5(3), 157-166. This complex quantitative inheritance was confirmed by several quantitative trait locus (QTL) analyses. This method allows for the mapping of polygenic inherited resistance and is a reliable technique for identifying the number and location of genetic resistance factors on the host plant's genetic linkage map. In this way, we were able to determine the multiple causative QTLs on each chromosome of sugar beet.

[0005] Mapping was performed using various circospora-resistant donors, but the observed QTL effect was mostly small. The maximum reported phenotypic effect was 5%.

[0006] In ongoing studies, lists of differentially expressed genes have been reported. A 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-microbe interactions, 24(7), 758-772) created genome-side expression profiles for various sugar beet genotypes (i.e., circospora-resistant, circospora-tolerant, circospora-sensitive, etc.) at the time of pathogen infection to analyze transcriptional changes in expression profiles related to leaf mottling. These analyses allowed the authors to create pathogen-induced transcriptional profiles for various sugar beet genotypes tested and identify potential candidate genes. However, these genes have not yet been characterized in detail. The genetic and functional background of circospora resistance, as well as the identity of resistance genes, have remained completely unknown until now.

[0007] However, quantitative inheritance of QTLs not only introduces desired resistance to Cercospora beticola into plants, but also often introduces undesirable traits, such as reduced yield, through the inheritance of additional genes associated with the positive traits of circospora resistance. This phenomenon is also known by the term "linkage drug." Furthermore, enormous breeding costs are required to select multiple resistance loci without reducing yield, which can negatively impact plant vitality. See Weiland and Koch, 2004.

[0008] Breeding companies have been offering circospora-resistant cultivars to the market for over a decade. The resistance of these cultivars is inherited through multiple resistance genes with limited effect. However, due to their complex genetic makeup, cultivar development is extremely difficult and complex, and such cultivars have the disadvantage of significantly lower yields compared to normal cultivars in the absence of invasiveness. In particular, this can be attributed to epigenetic interactions between several resistance genes and genes involved in sugar production, resulting in reduced plant fitness in the absence of the pathogen. Furthermore, circospora tends to overcome the resistance of cultivars established quite some time ago. Moreover, because basic research was conducted under different environmental conditions, different invasive pressures, and different circospora pathogen lines, the resistance scores available for previously unsuited wild genetic resources are usually unreliable and cannot be compared to one another. In this regard, it should be noted that environmental parameters such as humidity, temperature, and wind (which tend to be unstable) have a significant impact on the progression of circospora disease after infection. It is common for certain genetic resources to exhibit high levels of tolerance / resistance in one study and complete susceptibility in another. While there is a strong need for genes that can be easily introduced into existing cultivars and varieties to establish resistance to circospora, the aforementioned factors have made it impossible to identify dominant resistance genes that significantly impact circospora.

[0009] For example, novel breeding techniques based on gene editing using TALE nucleases or CRISPR systems, and the use of transgenic approaches, cannot be applied to currently available genetic material due to the complex genetics and the large number of genes involved in resistance expression, most of which have not yet been identified and characterized.

[0010] To counter the danger posed by Cercospora variants that overcome resistance, and for sustainable breeding against Cercospora leaf variegation, it is necessary to continuously identify new resistance genes and incorporate them into the gene pool of cultivated plants such as sugar beets. In particular, the objective was to provide suitable resistance genes that, after expression in plants, already exhibit a very large dominant resistance effect against Cercospora beticola. According to the present invention, this objective is achieved by embodiments characterized in the claims and specification.

[0011] Summary of the Invention The present invention relates to a nucleic acid molecule that can confer resistance to Circospora, particularly the fungus Circospora beticola, in plants, especially the subspecies Beta vulgaris vulgaris. The polypeptide encoded by the nucleic acid molecule is thereby produced in the plant. The nucleic acid molecule, which produces polypeptides after expression, already itself provides a very large dominant resistance effect against Circospora beticola in the plant.

[0012] Furthermore, the present invention relates to circospora-resistant plants, plant cells, plant organs, plant tissues, plant parts, seeds, seed stocks, or plant offspring that contain nucleic acid molecules or portions thereof endogenously or by genetic modification. According to certain any embodiments, plants and their components obtained by essentially biological processes are excluded.

[0013] The present invention also encompasses methods for increasing resistance to Circospora in plants of the Beta vulgaris species, and methods for creating, identifying, and possibly selecting Circospora-resistant plants. The present invention also encompasses methods for monitoring the invasion of the pathogen Circospora beticola, as well as oligonucleotides as probes and primers for hybridization with nucleic acid molecules according to the present invention.

[0014] Therefore, the present invention relates to embodiments listed in the following items and described in the examples and drawings.

[0015] [1] A nucleic acid molecule encoding a polypeptide that can confer resistance to circospora in plants expressing the polypeptide, (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 3; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 2; (c) A nucleotide sequence containing a DNA sequence selected from the group consisting of Sequence ID No. 1 or Sequence ID No. 53; (d) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence according to (a), (b), or (c); (e) A nucleotide sequence that encodes a polypeptide 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; (f) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 70% identical to the amino acid sequence of Sequence ID No. 3; (g) A nucleotide sequence that is at least 70% identical to the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 2 Includes a nucleotide sequence selected from, Resistance to Cercospora is preferably resistance to Cercospora beticola, or The plant is preferably a subspecies of Beta vulgaris subsp. vulgaris, and particularly preferably a sugar beet. A nucleic acid molecule characterized by the following features.

[0016] [2] A nucleic acid molecule according to [1], characterized in that the resistance effect against circospora conferred by the polypeptide is dominant in plants, preferably the polypeptide confers a resistance effect of at least 1 evaluation score, preferably 2 evaluation scores or more, particularly preferably at least 2 evaluation scores, particularly preferably at least 3 evaluation scores, and particularly preferably at least 4 evaluation scores.

[0017] [3] A nucleic acid molecule according to [1] or [2], characterized in that the nucleic acid molecule is derived from Beta vulgaris subsp. maritima.

[0018] A polypeptide encoded by a nucleic acid molecule from one of [4][1] to [3].

[0019] A vector or expression cassette comprising a nucleic acid molecule according to one of [5][1] to [3], wherein the nucleic acid molecule is preferably heterogeneous to the vector or expression cassette.

[0020] A cell comprising a nucleic acid molecule according to one of [6][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 gene or a transgene.

[0021] [7]In a Cercospora-resistant plant or a part thereof, comprising a nucleic acid molecule or nucleotide sequence according to one of [1] to [3], or a vector or expression cassette according to [5], wherein the plant that endogenously contains the nucleic acid molecule is of the Beta vulgaris species (however, not Beta vulgaris subsp. maritima), or a plant of Beta vulgaris subsp. vulgaris, a Cercospora-resistant plant or a part thereof, characterized in that. The nucleic acid molecule or nucleotide sequence can be contained endogenously or by genetic transformation. Seeds of the plant according to this paragraph can be obtained under accession number NCIMB 43646 from the deposit at NCIMB in Aberdeen, UK. The plant according to this paragraph can be obtained from the deposited seeds.

[0022] [8]A plant according to [7], characterized in that the plant is a hybrid plant.

[0023] [9]A plant according to [7] or [8], characterized in that the nucleic acid molecule is present heterozygously or homozygously in the genome of the plant.

[0024]

[10] A seed or progeny of a plant according to one of [7] to [9], comprising a nucleic acid molecule or nucleotide sequence according to one of [1] to [3], or a vector or expression cassette according to [5]. The nucleic acid molecule can be present by genetic transformation or non-genetic transformation or endogenously. Seeds according to this paragraph can be obtained under accession number NCIMB 43646 from the deposit at NCIMB in Aberdeen, UK. <

[0025]

[11] A method for increasing resistance to Cercospora in a plant, comprising the following steps: (i) Incorporating 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 homologous recombination repair or homologous recombination, preferably homologous recombination repair or homologous recombination supported by a site-specific nuclease, and optionally regenerating a plant from at least one plant cell; or (ii) In at least one cell of a plant, modifying preferably a native promoter, such as a native promoter comprising the DNA sequence according to SEQ ID NO: 7, or ligating a nucleic acid molecule according to one of [1] to [3] to a heterologous promoter having a higher activity level compared to a native promoter, such as a native promoter comprising the DNA sequence according to SEQ ID NO: 7, especially after Cercospora infection, to increase the expression of the nucleic acid molecule according to one of [1] to [3], and optionally regenerating a plant from at least one plant cell; or (iii) In at least one cell of a plant, increasing the activity and / or stability of the polypeptide according to [4] by modification of the nucleotide sequence of the nucleic acid molecule according to one of [1] to [3], and optionally regenerating a plant from at least one plant cell; or (iv) Transforming a plant cell with a nucleic acid molecule according to one of [1] to [3], or a vector or expression cassette according to [5], and optionally regenerating a (transgenic) plant from the transformed plant cell comprising, wherein the resistance to Cercospora is preferably resistance to Cercospora beticola, or the plant is preferably a plant of the Beta vulgaris species, preferably Beta vulgaris subsp. vulgaris, especially sugar beet, method.

[0026]

[12] A method for producing a Cercospora-resistant plant according to one of [7] to [9], comprising the following steps: (a) a step of transforming plant cells with a nucleic acid molecule from one of [1] to [3], or with a vector or expression cassette from [5]; and (b) the process of regenerating a transgenic plant from transformed plant cells; or (i) A step of introducing a site-specific nuclease and a repair matrix into cells of the plant species Beta vulgaris, wherein the site-specific nuclease can generate at least one double-strand break in the DNA in the cell's genome, preferably upstream and / or downstream of the target region, and the repair matrix comprises a nucleic acid molecule from one of [1] to [3]; (ii) A step of culturing cells from (i) under conditions that enable homologous recombination repair or homologous recombination, wherein nucleic acid molecules are incorporated from the repair matrix into the plant genome; and (iii)(ii) The process of regenerating plants from cells modified in (iii)(ii) Methods that include...

[0027]

[13] The method according to

[12] , characterized in that the target region comprises an allele variant of a nucleic acid molecule comprising one of [1] to [3], wherein the allele variant encodes a polypeptide that does not confer resistance to circospora or confers only slight resistance.

[0028]

[14] The method according to

[12] or

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

[13] .

[0029]

[15] Allele variants of nucleic acid molecules, (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 6; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 5; (c) Nucleotide sequence containing DNA sequence according to Sequence ID No. 4; (d) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence specified by (a), (b), or (c); (e) A nucleotide sequence that encodes a polypeptide different from the polypeptide encoded by the nucleotide sequence of (a), (b), or (c) by substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence; or (f) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 6. A method according to

[12] or

[13] , characterized by comprising a nucleotide sequence selected from.

[0030] A plant or part thereof that can be obtained or obtained by one of the methods of

[16]

[12] ~

[15] .

[0031]

[17] A method for identifying and optionally providing plant species of Beta vulgaris that are resistant to circospora, comprising at least step (i) or (ii): (i) a step of detecting the presence and / or expression of a nucleic acid molecule by one of [1] to [3] or the presence of a polypeptide by [4] in a plant or part of a plant, and / or (ii) A step of detecting at least one marker gene locus in the nucleotide sequence or cosegregation region of a nucleic acid molecule by one of [1] to [3]; and (iii) If applicable, the step of selecting Cercospora beticola-resistant plants. Includes, A method characterized in that detection in step (i) or step (ii) is based on the use of at least one molecular marker.

[0032]

[18] A method for identifying nucleic acid molecules encoding polypeptides that can confer resistance to circospora in plants of the species Beta vulgaris in which polypeptides are expressed, (i) Comparing the amino acid sequence of the polypeptide according to [4] with the amino acid sequence from a sequence database, or identifying allele variants encoding the polypeptide according to [4] in the genotype of the Beta vulgaris species; (ii) A step of identifying an amino acid sequence or an allele 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]; (iii) the step of introducing a nucleic acid molecule or allele mutant encoding an identified amino acid sequence into a Beta vulgaris plant to express the nucleic acid molecule in the plant; and (iv) Steps for detecting resistance to circospora A method characterized by including

[0033]

[19] A method for cultivating the plant of the species Beta vulgaris, (i) Prepare plants by one of [7] to [9], sow pelletized seeds of sugar beet plants or plants of the genus Bhutella by one of

[26] to

[39] , produce plants of the species Beta vulgaris using one of

[12] to

[15] , or identify and select plants of the genus Bhutella using the method of

[17] , and (ii) Cultivating plants or their offspring from (i) Includes, A method to prevent circospora from invading cultivated plants.

[0034] Oligonucleotides having 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 particularly preferably at least 100, 200, 300, or 500 nucleotides, that hybridize with a nucleotide sequence defined by one of

[20] [1] to [3].

[0035]

[21] Oligonucleotides, preferably pairs of oligonucleotides according to

[20] , or kits comprising these oligonucleotides, wherein the oligonucleotides are suitable as forward and reverse primers for hybridization with regions in the Beta vulgaris genome having circospora resistance conferred by polypeptides according to [4] in Beta vulgaris or co-separation with nucleic acid molecules according to one of [1] to [3].

[0036]

[22] Use of nucleic acid molecules by one of [1]-[3] in the creation of circospora-resistant plants of the subspecies Beta vulgaris vulgaris.

[0037]

[23] [1] Mutant variants, and / or (a) Sequence ID 7, (b) A nucleotide sequence that hybridizes under stringent conditions with a sequence complementary to the sequence in (a), (c) A nucleotide sequence that is at least 70% identical to the sequence shown in Sequence ID No. 7 Mutant promoters containing nucleic acid sequences selected from A method for creating an organism containing, (i) A step of preparing an organism or cell containing nucleic acid molecules and / or promoters, (II) A process of increasing the mutation rate of an organism or cell, or of inducing mutagenesis in an organism or cell. (III) A step of performing phenotypic selection of organisms that exhibit a change in resistance or a change in resistance level to Cercospora beticola as a result of mutation, or a step of performing genotypic selection of organisms or cells that include the mutations produced by step (II) in nucleic acid molecules and / or promoters. In addition, optionally, (IV) A process for regenerating an organism from the cells obtained in step (III). Methods that include...

[0038]

[24] The method by which the organism is a plant,

[23] .

[0039]

[25] The method according to

[24] , wherein the plant is Beta vulgaris, preferably Beta vulgaris subsp. vulgaris, more preferably sugar beet or red beet.

[0040] Pelleted seeds of sugar beets or chard plants, including red beets, containing nucleic acid molecules, as described in

[26] [1].

[0041]

[27] Beet bodies suitable for use as raw materials for industrial sugar production or for consumption as food,

[26] pelletized seeds.

[0042]

[28] Pelleted seeds according to

[26] or

[27] , wherein the pelletized seeds are monoembryonic seeds.

[0043]

[29] Pelleted seeds of sugar beet plants that can be harvested before bolting, according to

[26] ~

[28] .

[0044]

[30] Pelleted seeds according to

[26] -

[29] , where resistance to Cercospora is resistance to Cercospora beticola.

[0045]

[31] Pelleted seeds of sugar beet plants, which are biennials,

[26] ~

[30] .

[0046]

[32] Technical processing is performed, and the technical processing is (a) polishing; (b) Powder clothing; (c) forming the outer covering; and (d) Coloring Pelleted seeds selected from the group consisting of

[26] ~

[31] and

[0124] .

[0047]

[33] Pellets, (a) Insecticides; (b) Fungicides; and (c) Fertilizer Pelleted seeds according to

[26] -

[32] , comprising at least one chemical selected from a group selected from

[26] -

[32] .

[0048]

[34] Pelleted seeds according to

[26] -

[33] , in which the seeds are subjected to priming or pre-germination before or during pelletizing.

[0049]

[35] Pelleted seeds of sugar beet plants, hybrid sugar beet plants,

[26] ~

[34] .

[0050]

[36] Pelleted seeds according to

[26] -

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

[0051]

[37] Pelleted seeds by

[36] in which at least one mutation is a mutation of sequence number 1 or sequence number 2.

[0052]

[38] Pelleted seeds according to

[36] or

[37] , wherein the nucleotide sequence containing at least one mutation encodes a polypeptide having an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0053]

[39] Pelleted seeds according to

[38] , wherein the nucleotide sequence containing at least one mutation encodes a polypeptide having the amino acid sequence according to SEQ ID NO: 3.

[0054] A packing comprising pelletized seeds according to

[40]

[26] ~

[39] or a seed stock comprising nucleic acid molecules according to [1], wherein the preferred seed stock is a seed stock of a plant of the genus Chamaecyparis.

[0055]

[41] A mixture of pellets and sugar beet seeds, wherein the sugar beet seeds contain a nucleic acid sequence encoding a polypeptide that can confer resistance to circospora, and the nucleotide sequence is (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 3; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 2; (c) A nucleotide sequence containing a DNA sequence selected from the group consisting of Sequence ID No. 1 or Sequence ID No. 53; (d) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence according to (a), (b), or (c); (e) A nucleotide sequence that encodes a polypeptide 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; (f) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 70% identical to the amino acid sequence of Sequence ID No. 3; (g) A nucleotide sequence that is at least 70% identical to the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 2 A mixture selected from the group consisting of the following.

[0056]

[42] a) A step of preparing sugar beet plant seeds comprising a nucleic acid sequence encoding a polypeptide that can confer resistance to circospora, wherein the nucleotide sequence is (i) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 3; and (ii) A nucleotide sequence that is at least 95% identical to the DNA sequence by Sequence ID No. 1 or Sequence ID No. 2. A process selected from the group consisting of, b) The process of embedding sugar beet seeds into a pellet mass. c) A process of air-drying the pellet mass or drying the pellet mass. A method for producing pelletized sugar beet plant seeds, including

[26] ~

[39] .

[0057]

[43] Pelleted seeds having a nucleotide sequence containing at least one mutation, which is an artificial nucleotide sequence not found in nature,

[36] -

[39] .

[0058]

[44] The method according to

[23] , which involves applying a mutagenic chemical such as an EMS or mutagenic radiation during step (II).

[0059] A variety or cultivar of the genus Betel containing nucleic acid molecules according to

[45] [1], or pelletized seeds of said variety or cultivar.

[0060]

[46] [1] Leaves of Swiss chard plant containing nucleic acid molecules.

[0061] A bag containing one or more leaves of a chard plant, preferably a plastic bag, according to

[47]

[46] .

[0062]

[48] ​​A method for identifying plants resistant or tolerant to Circospora, preferably plants of the species Beta vulgaris or the subspecies Beta vulgaris vulgaris, (i) the step of detecting the presence and / or expression of nucleic acid molecules or nucleotide sequences defined in [1] in a plant or part of a plant, or the presence of polypeptides encoded by nucleic acid molecules or nucleotide sequences defined in [1]; and / or (ii) A step of detecting the nucleotide sequence of a nucleic acid molecule or at least one marker gene locus in a cosegregated region according to [1]. Includes, The co-separated region is a genomic region that co-separates from nucleic acid molecules or nucleotide sequences, conferring circospora resistance by polypeptides. The co-separation region preferably includes markers s4p1395s01 and s4p0421s01 and adjacent to them. A method characterized by the following: Preferably, the co-separation regions are sxi0123s02 and s4p0238s01, s4p1396s01 and sxh1195s02, s4p1398s01 and s4p0234s01, s4p1462s01 and s4p0232s01, s4p1464s01 and s4p0323s01, s4p0044s01 and s4p0322s01, s4p0056s01 and s4p0320s01, s4p0058s01 and sxh0942s04, s4p0059s Includes pairs of markers selected from the list consisting of 01 and sxh1834s05, s4p1564s01 and s4p0317s01, s4p1998s01 and s4p4308s01, s4p1343s01 and s4p4305d01, s4p1408s01 and sxh0678s01, s4p1565s01 and s4p4301s01, s4p1409s01 and s4p8772s01, s4p1411s01 and s4p4295s01, and adjacent to them. Most preferably, the co-separation regions are s4p1343s01 and s4p0421s01, s4p1408s01 and s4p0238s01, s4p1565s01 and sxh1195s02, s4p1409s01 and s4p0234s01, s4p1411s01 and s4p0232s01, s4p1414s01 and s4p0323s01, s4p1485s01 and s4p0322 It includes pairs of markers selected from the list consisting of s01, s4p0257s01 and s4p0320s01, s4p0258s01 and sxh0942s04, s4p0260s01 and sxh1834s05, sxh0876s05 and s4p0317s01, s4p0262s01 and s4p4308s01, s4p0263s01 and s4p4305d01, and adjacent to them. For mapping purposes, the following marker pairs are recommended: s4p4293s01 and s4p8772s01, preferably s4p4295s01 and s4p8772s01. The structural characteristics of the markers are shown in Table 1B. The method is as follows: - A step of preparing at least one plant, its tissue, seed or at least one cell thereof, - A step of extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed or at least one cell thereof, and -A step in which detection is performed on the extracted DNA. - The process of selecting circospora-resistant plants. It may be accompanied by one or more of the following.

[0063]

[49] The method involves further steps: - The process of creating electronically transmittable and / or electronically storable data representing the detection of the presence of nucleic acid molecules or nucleotide sequences. A method comprising

[48] or any one of

[0104] to

[0113] , characterized by including the above.

[0064]

[50] The method involves further steps: - The process of saving data to a computer-readable medium. A method according to

[49] , characterized by including the following:

[0065]

[51] A method by one of

[48] -

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

[0066]

[52] A process for identifying plants that exhibit resistance or tolerance to Circospora, comprising detecting at least one polymorphism, preferably a single nucleotide polymorphism, in the plant by at least one marker, wherein at least one marker is located in a chromosomal segment adjacent to two markers s4p1395s01 and s4p0421s01, or in a chromosomal segment adjacent to a pair of markers disclosed in

[48] , wherein the plant comprises a nucleic acid molecule or nucleotide sequence according to [1]. The markers may be diagnostic markers and / or molecular markers. The process comprises the following steps: - A process of preparing at least one plant, its tissue, seed or at least one cell thereof. - A step of extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed or at least one cell thereof. - A step of performing detection on the extracted DNA, and - The process of selecting circospora-resistant plants. It may include one or more of the following.

[0067]

[53] (i) At least one of the above single nucleotide polymorphisms is genetically linked to the nucleic acid molecule or nucleotide sequence according to [1], or has a recombination frequency of about 10%, preferably 5%, more preferably 1%, or less with respect to the nucleic acid molecule or nucleotide sequence according to [1], (ii) At least one of the above single nucleotide polymorphisms is located within the range of 2562kbp, 2300kbp, 2100kbp, 1900kbp, 1700kbp, 1500kbp, 1300kbp, 1100kbp, 900kbp, 700kbp, 500kbp, 300kbp, 100kbp, 50kbp, 25kbp, 10kbp, 5kbp, or 1kbp or less from the nucleic acid molecule or nucleotide sequence according to [1]. The process described in

[52] .

[0068]

[54] A process or method by any one of

[0104] to

[0113] that causes a plant to exhibit increased resistance or tolerance to circospora compared to a plant lacking the nucleic acid molecule or nucleotide sequence of [1].

[48] ,

[52]

[0069]

[55] A single nucleotide polymorphism is a process by which any one of the following nucleotides is genetically and / or statistically associated with the presence of a nucleic acid molecule or nucleotide sequence by [1]

[52] -

[54] ,

[57] -

[70] ,

[0106] -

[0113] .

[0070]

[56] A process according to

[52] -

[55] , wherein at least one marker is one of the markers shown in Table 1B, or at least one marker is one of the marker pairs disclosed in

[48] .

[0071]

[57] A process for identifying plants that are resistant or tolerant to circospora, comprising detecting at least two single nucleotide polymorphisms in the plant or in the DNA of the plant by at least two markers, at least one of the markers is s4p1395s01, sxi0123s02, s4p1396s01, s4p1398s01, s4p1462s01, s4p1464s01, s4p0044s01, s4p00 56s01, s4p0058s01, s4p0059s01, s4p1564s01, s4p1998s01, s4p1343s01, s4p1408s01, s4p1565s01 , s4p1409s01, s4p1411s01, s4p1414s01, s4p1485s01, s4p0257s01, s4p0258s01, s4p0260s01, sxh0 876s05, s4p0262s01, s4p0263s01, s4p0264s01, s4p2271s01, s4p4288s01, s4p4290d01, s4p4293s01 A marker selected from the group consisting of [1] and a nucleotide sequence according to [1] are located on or within an adjacent first chromosome segment, and at least one of the markers is a polynucleotide of claim 1, s4p0421s01, s4p0238s01, sxh1195s02, s4p0234s01, s4p0232s01, s4p0323s01, s4p03 22s01, s4p0320s01, sxh0942s04, sxh1834s05, s4p0317s01, s4p4308s01, s4p4305d01 A process in which a marker selected from a group consisting of the following is located on or within an adjacent second chromosome segment.

[0072]

[58] A process by

[57] in which at least two markers and a nucleotide sequence by [1] are adjacent to the first and second chromosomal segments without being part of the first or second chromosomal segment.

[0073]

[59] A process according to

[57] or

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

[0074]

[60] At least one marker is located in 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 preferably spans the entire length of SEQ ID NO: 74 or SEQ ID NO: 75, by the process of

[57] -

[59] .

[0075]

[61] at least one of two markers is located in a genomic region having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 74, and at least one of two markers is located in a genomic region having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with sequence number 75, wherein the identity preferably extends over the entire length of sequence number 74 or sequence number 75, as described in

[57] -

[60] .

[0076]

[62] A process according to

[57] -

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

[0077]

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

[57] -

[62] .

[0078]

[64] The process according to

[57] -

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

[0079]

[65] The following steps: - A step of preparing at least one plant, its tissue, seed or at least one cell thereof, - A step of extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed or at least one cell thereof, and - A step of performing a detection step on the extracted DNA as defined in any of

[57] to

[64] . The process includes

[57] ~

[64] .

[0080]

[66] A process or method according to

[48] -

[65] , wherein the detection step includes a polymerase chain reaction (PCR).

[0081]

[67] A process or method according to

[48] -

[65] or

[0112] or

[0113] , using polymerase chain reaction (PCR), wherein the PCR uses two allele-specific forward primers (or use thereof), and the detection step uses fluorescence resonance energy transfer (FRET), wherein the presence, absence, or type of fluorescence is determined by the sensor. The sensor signal can be converted into electronically transmittable and / or electronically storable data representing the detection of the presence of nucleic acid molecules or nucleotide sequences according to [1]. In addition, the electronically transmittable and / or electronically storable data can be stored on a computer-readable medium. The type of fluorescence may be its color / wavelength, or a specific dye involved in the fluorescence (e.g., FAM or HEX).

[0082]

[68] The process according to

[67] , which further involves the use of one common reverse primer or the use of one common reverse primer. Examples of common primers are shown in Table 1B.

[0083]

[69] The process is one of the following:

[67] ,

[68] ,

[0112] , and

[0113] , in which the determination of fluorescence by the sensor is endpoint fluorescence reading.

[0084]

[70] A process by which each of two allele-specific forward primers contains a unique tail sequence that is chemically bound to a specific FRET cassette during PCR,

[67] or

[69] ,

[0112] and

[0113] .

[0085]

[71] A method for identifying plants resistant or tolerant to Circospora, preferably of the species Beta vulgaris or the subspecies Beta vulgaris vulgaris, comprising the following steps: - A step of preparing at least one plant, its tissue, seed or at least one cell thereof, - A step of extracting DNA, preferably genomic DNA, from at least one plant, its tissue or at least one cell, and -A step of detecting the presence or absence of nucleic acid molecules or nucleotide sequences in the extracted DNA by [1], preferably by markers shown in Table 1B, or marker pairs disclosed in

[48] , oligonucleotide pairs by

[20] ,

[21] ,

[72] or

[73] or

[76] , a set of three oligonucleotides by

[74] or

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

[84] , -Optionally, the step of selecting plants in which the extracted DNA contains nucleic acid molecules or nucleotide sequences according to [1]. A method characterized by including

[0086]

[72] A pair of oligonucleotides suitable for use as primers in PCR, comprising the markers s4p1395s01 and s4p0421s01 and capable of hybridizing to adjacent genomic segments. Preferably, the genomic segments are sxi0123s02 and s4p0238s01, s4p1396s01 and sxh1195s02, s4p1398s01 and s4p0234s01, s4p1462s01 and s4p0232s01, s4p1464s01 and s4p0323s01, s4p0044s01 and s4p0322s01, s4p0056s01 and s4p0320s01, s4p0058s01 and sxh0942s04, s4p0059s0 Includes pairs of markers selected from the list consisting of 1 and sxh1834s05, s4p1564s01 and s4p0317s01, s4p1998s01 and s4p4308s01, s4p1343s01 and s4p4305d01, s4p1408s01 and sxh0678s01, s4p1565s01 and s4p4301s01, s4p1409s01 and s4p8772s01, s4p1411s01 and s4p4295s01, and adjacent to them. Most preferably, the genome segments are s4p1343s01 and s4p0421s01, s4p1408s01 and s4p0238s01, s4p1565s01 and sxh1195s02, s4p1409s01 and s4p0234s01, s4p1411s01 and s4p0232s01, s4p1414s01 and s4p0323s01, s4p1485s01 and s4p0322 The genomic interval includes and is adjacent to a pair of markers selected from the list consisting of s01, s4p0257s01, s4p0320s01, s4p0258s01, sxh0942s04, s4p0260s01, sxh1834s05, sxh0876s05, s4p0317s01, s4p0262s01, s4p4308s01, s4p0263s01, and s4p4305d01. The genomic interval may be a genomic interval of the plant species Beta vulgaris or the subspecies Beta vulgaris subsp. vulgaris.

[0087]

[73] A pair of oligonucleotides suitable for use as primers in PCR, which can hybridize the nucleic acid sequence of SEQ ID NO: 74 or SEQ ID NO: 75, or the entire length of the sequence of SEQ ID NO: 74 or SEQ ID NO: 75, to a sequence that is at least 99% identical to the sequence of SEQ ID NO: 74 or SEQ ID NO: 75.

[0088]

[74] A set of three oligonucleotides suitable for use as primers in PCR, comprising two forward primers and a reverse primer, wherein each primer has a different nucleotide sequence, and only the reverse primer and one of the two forward primers can hybridize to the markers s4p1395s01 and s4p0421s01 and adjacent genomic segments. Preferably, the genome segments are sxi0123s02 and s4p0238s01, s4p1396s01 and sxh1195s02, s4p1398s01 and s4p0234s01, s4p1462s01 and s4p0232s01, s4p1464s01 and s4p0323s01, s4p0044s01 and s4p0322s01, s4p0056s01 and s4p0320s01, s4p0058s01 and sxh0942s04, s4p0059s0 Includes pairs of markers selected from the list consisting of 1 and sxh1834s05, s4p1564s01 and s4p0317s01, s4p1998s01 and s4p4308s01, s4p1343s01 and s4p4305d01, s4p1408s01 and sxh0678s01, s4p1565s01 and s4p4301s01, s4p1409s01 and s4p8772s01, s4p1411s01 and s4p4295s01, and adjacent to them. Most preferably, the genome segments are s4p1343s01 and s4p0421s01, s4p1408s01 and s4p0238s01, s4p1565s01 and sxh1195s02, s4p1409s01 and s4p0234s01, s4p1411s01 and s4p0232s01, s4p1414s01 and s4p0323s01, s4p1485s01 and s4p0322 It includes pairs of markers selected from the list consisting of s01, s4p0257s01 and s4p0320s01, s4p0258s01 and sxh0942s04, s4p0260s01 and s4h1834s05, sxh0876s05 and s4p0317s01, s4p0262s01 and s4p4308s01, s4p0263s01 and s4p4305d01, and is adjacent to them.

[0089]

[75] A set of three oligonucleotides suitable for use as primers in PCR, comprising two forward primers and a reverse primer, wherein each primer has a different nucleotide sequence, and only the reverse primer and one of the two forward primers can hybridize to the sequence of SEQ ID NO: 74 or SEQ ID NO: 75, or to a sequence that is at least 99% identical to the sequence of SEQ ID NO: 74 or SEQ ID NO: 75 over its entire length.

[0090]

[76] A pair or set of oligonucleotides according to

[72] -

[75] , each oligonucleotide comprising 15-40 nucleotides, preferably 17-30 nucleotides, more preferably 19-25 nucleotides.

[0091]

[77] A process for quantifying the level of resistance or tolerance of a plant to Circospora, comprising the following steps: - A step of preparing at least one first plant or at least one seed of the first plant containing nucleic acid molecules or nucleotide sequences according to [1], and at least one second plant or at least one seed of the second plant lacking nucleic acid molecules or nucleotide sequences according to [1], - A process of cultivating plants or germinating seeds and cultivating germinated plants, wherein a) cultivation conditions allow for interaction with Circospora, or b) inoculating plants with Circospora. - A step of determining the damaged surface of at least one leaf of each of two plants, -Optionally, a step of comparing the resistance or tolerance level of the first plant with the resistance or tolerance level of the second plant, and / or determining an evaluation score for the first and / or second plant selected from Table 1A. A process that includes this.

[0092]

[78] A process according to

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

[0093]

[79] A method for reducing the application of fungicides, comprising the following steps: I) A step of preparing seeds or seed stocks containing nucleic acid molecules or nucleotide sequences according to [1], II) The process of sowing or rowing seeds or seed stock in order to enable germination of seeds or seed stock, III) A process of cultivating the germinated plants obtained in step II), IV) A process to harvest the plants cultivated in step III), or to harvest plant parts such as storage organs or taproots, or to harvest the seeds of the plants cultivated in step III). A method that includes reducing or avoiding the application of fungicides during step II) and / or step III).

[0094]

[80] The method according to

[79] , wherein the fungicide is an effective fungicide against Circospora, and these fungicides may include one or more fungicides effective against Circospora, such as the following fungicides: epoxyconazole, kresoximmethyl, thiophanatemethyl, mancozeb, and others referred to in other parts of this specification.

[0095]

[81] The method according to

[79] or

[80] wherein the application of fungicides in step II) and / or step III) is reduced or avoided compared to the cultivation of nucleic acid molecule-deficient plants by [1] under the same or equivalent conditions as the plants in step III).

[0096] Use of markers or molecular markers for the identification and / or selection of plants containing nucleic acid molecules or nucleotide sequences

[82] [1].

[0097]

[83] Use by a marker or molecular marker in a process or method according to

[48] -

[71] ,

[82] .

[0098] A molecular marker, or preferably a set of molecular markers comprising two or three molecular markers, suitable for use in processes or methods according to

[84]

[48] ~

[71] .

[0099] A method for expressing a nucleic acid molecule or nucleotide sequence according to

[85] [1], comprising the following steps: i) A step of introducing nucleic acid molecules or nucleotide sequences into one or more cells according to [1], ii) The step of culturing one or more cells under conditions that enable the proliferation of one or more cells and / or enable the expression of nucleic acid molecules or nucleotide sequences by [1]. Methods that include...

[0100]

[86] The method is for in vitro expression, as described in

[85] .

[0101]

[87] The method according to

[85] or

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

[0102]

[88] Methods according to

[85] -

[87] , in which the introduction of nucleic acid molecules or nucleotide sequences is the introduction of a vector, plasmid, or transfer DNA (tDNA).

[0103]

[89] The method according to

[85] -

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

[0104]

[90] The method according to

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

[0105]

[91] A method according to

[90] in which a 35S promoter derived from cauliflower mosaic virus comprises or consists of the nucleic acid sequence of sequence number 225.

[0106]

[92] The method according to

[85] -

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

[0107]

[93] A method according to

[85] -

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

[0108]

[94] The plant further comprises a nucleic acid molecule encoding a polypeptide that can induce resistance to the pathogen beet necrotic yellow vein virus BNYVV in the plant expressing the polypeptide, the nucleic acid molecule a) A nucleotide sequence encoding a polypeptide having the amino acid sequence specified by SEQ ID NO. 227 or SEQ ID NO. 228, b) A nucleotide sequence containing the coding sequence of the DNA sequence according to SEQ ID NO: 226, c) A nucleotide sequence that hybridizes under stringent conditions with the complementary sequence of the nucleotide sequence according to a) or b), d) A nucleotide sequence encoding a polypeptide obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence encoded by the nucleotide sequence according to a) or b), e) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 90%, preferably at least 95%, most preferably 99%, identical to the amino acid sequence encoded by the nucleotide sequence according to a) or b), or f) A nucleotide sequence encoding at least one nucleotide-binding domain (NBS) corresponding to amino acid positions 168-227 of SEQ ID NO: 227 or 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 605-627 of SEQ ID NO: 228, and / or at least one internal repeat domain (IR) corresponding to amino acid positions 1013-1072 of SEQ ID NO: 227 or 1027-1086 of SEQ ID NO: 228. A plant according to [7]-[9], characterized by containing a nucleotide sequence selected from [7]-[9].

[0109]

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

[0110]

[96] Seeds or offspring can be obtained from those deposited at the NCIMB in Aberdeen, England, with access number NCIMB 43646,

[10] Seeds or offspring.

[0111]

[97] Plants according to [7]-[9],

[94] and

[95] , further characterized by the plant containing sequence number 182 or being detectable by the marker s4p8772s01.

[0112]

[98] A process for breeding circospora-resistant Beta vulgaris species, comprising the following steps: i) The process of obtaining seeds from those deposited at the NCIMB in Aberdeen, UK, under access number NCIMB 43646, or from the descendants of deposited seeds. ii) The process of cultivating the seeds from process i) under conditions that allow for plant growth, iii) A process of crossbreeding the plants obtained from step ii) with plants of the Beta vulgaris species. A process that includes this. The method is optional. iv) A further step of determining the presence or absence of nucleic acid molecules or nucleotide sequences in the offspring obtained from step iii) by the method or process described herein, for example, by the methods or processes of

[48] to

[71] . It may include.

[0113]

[99] Use of electronically transmittable and / or electronically stowable data obtained by any one of the processes of

[49] ,

[50] ,

[69] ,

[70] and

[0104] to

[0113] for determining the estimated breeding value of plants in a population of plants.

[0114]

[0100] Use by

[99] in which the estimated breeding value depends at least in part on circospora resistance and the plant population includes 1000 or fewer plants.

[0115]

[0101] A plant by one of [7] to [9] or a seed or offspring of a plant by

[10] , which contains or incorporates a nucleic acid molecule or nucleotide sequence by [1] as genetic transfer.

[0116]

[0102] A plant storage organ according to one of [7]~[9] or

[0101] . The storage organ may be the taproot, especially the beet root.

[0117]

[0103] A single or multiple leaf of a plant according to one of [7]~[9] or

[0101] , wherein the plant may be Swiss chard or a plant of the genus Spinach.

[0118]

[0104] A method for identifying plants that are resistant or tolerant to Circospora, (i) (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 3; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 2; (c) A nucleotide sequence containing the DNA sequence by Sequence ID No. 1 or Sequence ID No. 53; (d) Nucleotide sequences that hybridize with the complementary sequence of the nucleotide sequence by (a), (b), or (c) by repeated washing in 4×SSC (physiological saline-sodium citrate) at 65°C, followed by washing in 0.1×SSC at 65°C for a total of approximately 1 hour; (e) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 3; (f) A nucleotide sequence that is at least 90% identical to the DNA sequence by Sequence ID No. 1 or Sequence ID No. 2. A step of detecting the presence or absence of a nucleotide sequence selected from the group consisting of the following; (ii) A step of detecting the presence or absence of a polypeptide encoded by the nucleotide sequence defined in step (i) in a plant or part of a plant; and / or (iii) A step to detect at least one marker gene locus in the nucleotide sequence or cosegregation region defined in step (1). Includes, The co-separated region is a genomic region that co-separates from nucleic acid molecules or nucleotide sequences, conferring circospora resistance by polypeptides. The co-separation region contains markers s4p1395s01 and s4p0421s01, and adjacent to them A method characterized by the following features.

[0119]

[0105] The method is (iv) Further steps to select circospora-resistant plants A method according to

[0104] , characterized by including

[0104] .

[0120]

[0106] A method according to

[0104] or

[0105] wherein the detection in step (i) or (iii) is based on at least one polymorphism or single nucleotide polymorphism.

[0121]

[0107] a) At least one of the above polymorphisms or single nucleotide polymorphisms is genetically linked to the nucleotide sequence or has a recombination frequency of 10% or less relative to the nucleotide sequence. b) At least one of the above polymorphisms or single nucleotide polymorphisms is located within the range of 2562kbp, 2300kbp, 2100kbp, 1900kbp, 1700kbp, 1500kbp, 1300kbp, 1100kbp, 900kbp, 700kbp, 500kbp, 300kbp, 100kbp, 50kbp, 25kbp, 10kbp, 5kbp, or 1kbp or less from the nucleotide sequence. c) At least one of the above polymorphisms or single nucleotide polymorphisms is detectable in seeds deposited at the NCIMB in Aberdeen, UK, with access number NCIMB 43646, or d) At least one of the above polymorphisms or single nucleotide polymorphisms is part of a cosegregation region. Includes, The nucleotide sequence is the nucleotide sequence defined in step (i) of

[0104] , The coseparation region is the coseparation region defined in step (iii) of

[0104] . The method according to

[0106] is further characterized by the fact that...

[0122]

[0108] The following steps are terminated by detection: a) A step of preparing at least one plant, its tissue, a seed or at least one cell thereof, and b) The step of extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed, or at least one cell thereof. A method that further includes any one of

[0104] to

[0107] . In other words, for example, A method for identifying plants that are resistant or tolerant to Circospora, comprising the following steps: ia) the process of preparing at least one plant, its tissue, seed or at least one of its cells, and ib) A step of extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed or at least one of its cells, and ic) A step to detect the presence or absence of the nucleotide sequence defined in step (i) of

[0104] , ii) A step of detecting the presence or absence of a polypeptide encoded by the nucleotide sequence defined in step (i) in a plant or part of a plant; and / or iiia) A step of preparing at least one plant, its tissue, seed or at least one of its cells, and iiib) Extracting DNA, preferably genomic DNA, from at least one plant, its tissue, seed, or at least one of its cells, and iiic) A step to detect at least one marker gene locus in the nucleotide sequence or cosegregation region defined in step (1). Includes, The co-separated region is a genomic region that co-separates from nucleic acid molecules or nucleotide sequences, conferring circospora resistance by polypeptides. The co-separation region contains markers s4p1395s01 and s4p0421s01, and adjacent to them A method characterized by the following features.

[0123]

[0109] The method according to

[0104] to

[0108] , involving the use of at least two oligonucleotides.

[0124]

[0110] The oligonucleotide is suitable for use as a primer in PCR and comprises the markers s4p1395s01 and s4p0421s01 and can hybridize to a genomic section adjacent to them or adjacent to the marker pair disclosed in

[48] by the method of

[0109] . Preferably, the oligonucleotide can hybridize in PCR to a genomic template comprising the nucleotide sequence defined in step (i) of

[0104] in a manner / distance such that the resulting amplification product comprises up to 2000 bp, preferably up to 1500 bp, more preferably 1000 bp, most preferably up to 500 or 200, or up to 100 bp.

[0125]

[0111] A method according to any one of the above claims, wherein the plant is a plant of the genus Betel chard, spinach, soybean, carrot, or angelica.

[0126]

[0112] A method according to any one of

[0104] to

[0111] , wherein PCR is used in steps (i) and / or (iii), the PCR uses two allele-specific forward primers, the detection step uses fluorescence resonance energy transfer, the presence, absence or type of fluorescence is determined by the sensor, and a sensor signal is optionally generated. The sensor signal can be converted into electronically transmittable and / or electronically storable data representing the detection of the presence of a nucleic acid molecule or nucleotide sequence according to [1]. In addition, the electronically transmittable and / or electronically storable data can be stored on a computer-readable medium. The determination of fluorescence by the sensor may be an endpoint fluorescence reading.

[0127]

[0113] The method according to

[0112] , further using one common reverse primer. Preferably, the reverse primer and allele-specific forward primer can hybridize with a genome template containing the nucleotide sequence defined in step (i) of

[0104] in a manner / distance such that the resulting amplified product contains up to 2000 bp, preferably up to 1500 bp, more preferably 1000 bp, most preferably up to 500 or 200, or up to 100 bp in PCR. The primers may be oligonucleotides, e.g., oligonucleotides referred to in

[0109] .

[0128]

[0114] In plants of the genera Betel chard, spinach, soybean, carrot, or angelica, the nucleic acid molecule encoding a polypeptide that can confer resistance to circospora in plants expressing the polypeptide, (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 3; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 2; (c) A nucleotide sequence containing the DNA sequence by Sequence ID No. 1 or Sequence ID No. 53; (d) Nucleotide sequences that hybridize with the complementary sequence of the nucleotide sequence according to (a), (b), or (c) by repeated washing in 4×SSC at 65°C and then in 0.1×SSC at 65°C for a total of approximately 1 hour; (e) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 3; (f) A nucleotide sequence that is at least 90% identical to the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 2 It includes a nucleotide sequence selected from the group consisting of, A plant of the genus Branthus or Spinach, further comprising an endogenous allele encoding an epsp synthase having an amino acid different from proline at position 179. According to a particular embodiment, this epsp synthase is a mutant epsp synthase, the mutation being generated by mutagenesis, radiation, gene editing, site-directed point mutation, zinc finger nuclease, TALEN, or subsequent selection by in vitro culture of the respective cells and the use of herbicides such as glyphosate.

[0129]

[0115] A plant by

[0114] that is further characterized by being a hybrid plant.

[0130]

[0116] A plant according to

[0114] or

[0115] , further characterized in that the plant is a doubling haploid plant.

[0131]

[0117] A plant comprising any one of

[0114] to

[0116] , further characterized by dominant resistance to Circospora.

[0132]

[0118] A plant according to any one of

[0114] to

[0117] , further characterized by the inclusion of nucleic acid molecules or nucleotide sequences as genetic transfer.

[0133]

[0119] A plant comprising any one of

[0114] to

[0118] , further characterized by being homozygous for nucleic acid molecules or nucleotide sequences.

[0134]

[0120] A plant according to any one of

[0114] to

[0119] , further characterized in that the plant has resistance to glyphosate.

[0135]

[0121] A plant comprising any one of

[0114] to

[0120] , further characterized by containing a nucleic acid molecule or nucleotide sequence as an introduced gene.

[0136]

[0122] A plant according to any one of

[0114] to

[0121] , further characterized in that a nucleic acid molecule or nucleotide sequence can be obtained from seeds deposited at the NCIMB in Aberdeen, UK, with access number NCIMB 43646.

[0137] A plant storage organ or leaf, one of the following:

[0123] ,

[0114] , or

[0122] .

[0138] A pelletized seed of a plant, wherein the seed contains nucleic acid molecules and endogenous alleles, in a manner that is consistent with any one of

[0124] ,

[0114] , to

[0122] .

[0139]

[0125] At position 179, there is an epsp synthase that has an amino acid different from proline. i) Array of sequence number 223, ii) Sequences having an amino acid different from serine and proline at position 179 i), or iii) A sequence that has at least 90% identity with the sequence in i) or ii) throughout its entire length. A plant comprising any one of

[0114] to

[0122] , containing an amino acid sequence selected from, a storage organ or leaf according to claim 13, or a pelletized seed according to claim 14.

[0140]

[0126] A method according to any one of

[0104] to

[0113] , wherein the cosegregation region is a cosegregation region defined in

[48] .

[0141]

[0127] A method according to any one of

[0109] to

[0113] , wherein at least two oligonucleotides are oligonucleotides defined in

[20] ,

[21] ,

[72] , or

[73] .

[0142]

[0128] A mixture of pelletized mass and sugar beet plant seeds, wherein the sugar beet plant seeds are seeds of a plant from any one of

[0114] to

[0122] , and the seeds contain nucleic acid molecules and endogenous alleles.

[0143] First, some of the terms used in this application will be explained in detail below: In the context of this invention, "evaluation score" is understood as a qualitative assessment of resistance to circospora invasion and is expressed using a scale of 1 to 9 (1 = strong resistance and 9 = no resistance).

[0144] [Table 1]

[0145] The genus Cercospora includes various species, such as Cercospora arachidicola, Cercospora ariminiensis, Cercospora asparagi, Cercospora bertoreae, Cercospora beticola, Cercospora bizzozeriana, Cercospora canescens, Cercospora carotae, Cercospora chenopodii, Cercospora cistinearum, and Cercospora cladospolioides. Cercospora cladosporioides), Cercospora diazu, Cercospora dulcamarae, Cercospora erysimi, Cercospora hayii, Cercospora kikuchii, Cercospora malvacearum, Cercospora malvicola, Cercospora medicaginis, Cercospora oryzaem, Cercospora personata, Cercospora plantaginis (Cercospora This includes species such as plantaginis, Cercospora ricinella, Cercospora setariae, Cercospora unamunoi, Cercospora violae, or Cercospora zeae-maydis.

[0146] With regard to specifying the length of a nucleotide sequence, the term "approximately" means a deviation of ±200 base pairs, preferably ±100 base pairs, and particularly preferably ±50 base pairs.

[0147] Cosegregated regions are genomic regions that are inherited in most cases, or always, together with genomic regions, loci, genes, polymorphisms, or single nucleotide polymorphisms that are genetically linked, genetically connected, or adjacent or nearby. For example, genetic linkage may be inherited by the next generation with at least 95%, 96%, 97%, 98%, or 99%, or cosegregated regions and genomic regions, loci, genes, polymorphisms, or single nucleotide polymorphisms that are genetically linked, genetically connected, or adjacent or nearby may be at distances of up to 2562kbp, 2300kbp, 2100kbp, 1900kbp, 1700kbp, 1500kbp, 1300kbp, 1100kbp, 900kbp, 700kbp, 500kbp, 300kbp, 100kbp, 50kbp, 25kbp, 10kbp, 5kbp, or 1kbp, or less. The presence of co-segregated regions suggests or diagnoses a corresponding genomic region, locus, gene, polymorphism, or single nucleotide polymorphism. Co-segregated regions can be detected / identified, for example, in seeds deposited at the NCIMB in Aberdeen, UK, under access number NCIMB 43646, by the corresponding methods, markers, and oligonucleotides contained herein. In connection with the present invention, co-segregated regions may be genomic regions in plants of the genera Brantia, Spinach, Glycine, Carrot, or Angelica.

[0148] "Plants" include, but are not limited to, plants of different genera such as Swiss chard, spinach, soybeans, carrots, and angelica.

[0149] The genus Beta belongs to the family Amaranthaceae. These plants include the species Beta macrocarpa, Beta vulgaris, Beta lomatogona, Beta macrorhiza, Beta corolliflora, Beta trigyna, and Beta nana. The species Beta vulgaris is particularly known as the subspecies Beta vulgaris subsp. vulgaris. For example, these include Beta vulgaris subsp. vulgaris var. altissima (sugar beet in the narrow sense), Beta vulgaris ssp. vulgaris var. vulgaris (chard), Beta vulgaris ssp. vulgaris var. conditiva (beetroot / red beet), and Beta vulgaris ssp. vulgaris var. crassa / alba (feed beet). It should be noted that the nucleic acids according to the present invention do not occur naturally in sugar beet, chard, beetroot, or feed beet, but can be artificially introduced into them.

[0150] Plants belonging to the genus Spinacia belong to the family Amaranthaceae. This genus specifically includes Spinacia oleracea, also known as spinach.

[0151] Plants belonging to the genus Glycine belong to the family Fabaceae. This genus specifically includes Glycine max, also known as soybean.

[0152] Plants of the genus Daucus belong to the family Apiaceae. This genus specifically includes Daucus carota and Daucus carota subsp. sativus, also known as carrot.

[0153] Plants of the genus *Pastinaca* belong to the family Apiaceae. This genus specifically includes *Pastinaca sativa*, also known as parsnip.

[0154] A "functional fragment" of a nucleotide sequence refers to a segment of the nucleotide sequence that has the same or equivalent functionality as the complete nucleotide sequence from which the functional fragment originates. Therefore, a functional fragment may have nucleotide sequences identical or homologous to the entire nucleotide sequence over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99%. The 90–100% range is also explicitly included. Furthermore, a "functional fragment" of a nucleotide sequence may also refer to a segment of the nucleotide sequence that modifies the functionality of the entire nucleotide sequence, for example, during post-transcriptional or transcriptional gene silencing. Therefore, the functional fragment of the nucleotide sequence may include at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the entire nucleotide sequence, preferably at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, or 140 consecutive nucleotides, particularly preferably at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 consecutive nucleotides. The range of 21 to 50 nucleotides is also explicitly included.

[0155] The “functional portion” of a protein means a segment of the protein or a portion of the amino acid sequence that codes for a protein, and such segment may exhibit the same or equivalent functionality as the complete protein in plant cells. The functional portion of a protein has an amino acid sequence that is identical to or similar to the protein from which it originates, taking into account conserved and semi-conserved amino acid exchanges, for at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99% of its length.

[0156] The term "heterogeneous" means that the introduced polynucleotide originates from a cell or organism with a different genetic background, either homogeneous or heterogeneous, or is homologous to a prokaryotic or eukaryotic host cell but differs from the naturally occurring corresponding polynucleotide due to being placed in a different genetic environment. Heterogeneous polynucleotides may be present in addition to the corresponding endogenous genes.

[0157] In the sense of the present invention, "homolog" is understood as a protein of the same phylogenetic origin, "analog" is understood as a protein that performs the same function but has a different phylogenetic origin, "ortholog" is understood as a protein of a different species that performs the same function, and "paralog" is understood as a protein that has appeared within a species due to duplication, and this copy either retains the same protein function, changes its expression template rather than its function, alters its protein function, or distributes the original gene function between the two copies.

[0158] "Hybridizing" or "hybridization" should be understood as the process by which a single-stranded nucleic acid molecule binds to the most complementary nucleic acid chain possible, i.e., forms base pairs 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 is preferable to understand "hybridizing" or "hybridization" as 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 forming base pairs with the most complementary nucleic acid chain possible. The possibility of such annealing depends on the stringency of the hybridization conditions. The term "stringency" refers to the hybridization conditions. High stringency exists when base pairing is more difficult, and 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 increasing the temperature and / or decreasing the salt content. "Stringent hybridization conditions" should be understood as conditions in which hybridization occurs mainly between homologous nucleic acid molecules. Thus, the term "hybridization conditions" refers not only to the conditions used when actually adding nucleic acids, but also to the conditions used in the subsequent washing process. For example, stringent hybridization conditions are those in which only 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.Stringent hybridization conditions include, for example, hybridization in 4×SSC at 65°C, followed by repeated washing in 0.1×SSC at 65°C for a total of approximately 1 hour. Hybridization is preferably carried out under stringent conditions.

[0159] A sequence that exhibits a certain level of identity with respect to a starting sequence can exhibit that given level of identity throughout the entire length of the starting sequence. This applies not only to amino acid sequences but also to nucleotide sequences.

[0160] With respect to nucleic acids in the form of double-stranded DNA, a “complementary” nucleotide sequence means that a second DNA strand complementary to the first DNA strand has nucleotides corresponding to the bases of the first strand, according to the rules of base pairing. The complementary sequence is preferably perfectly complementary to the opposite sequence and therefore preferably of the same length.

[0161] "Isolated nucleic acid molecules" are understood as nucleic acid molecules extracted from their natural or original environment. This term also includes synthetically produced nucleic acid molecules. "Isolated polypeptides" are understood as polypeptides extracted from their natural or original environment. This term also includes synthetically produced polypeptides.

[0162] A “molecular marker” or “marker” is a nucleic acid that is polymorphic in a plant population and is used as a reference or localization point. Depending on the technical context, the term “marker” may be associated with a specific genomic location detectable by the corresponding “molecular marker,” and “molecular markers” are usually sequentially fitted to genomic locations. Markers for detecting recombination events are preferably suitable for monitoring differences or polymorphisms within a plant population. For this reason, such markers can detect and distinguish various allele states (alleles). The term “molecular marker” also refers to nucleotide sequences that are complementary, or at least largely complementary, or homologous to the genomic sequence, for example, the nucleic acid used as a probe or primer. These differences at the DNA level should be found as markers, such as polynucleotide sequence differences, e.g., SSR (simple sequence repeat), RFLP (restriction fragment length polymorphism), FLP (fragment length polymorphism), or SNP (single nucleotide polymorphism). Markers may be derived from genomic nucleic acids or expressed nucleic acids, such as splice RNA, cDNA, or ESTs, and may be associated with nucleic acids that are suitable for use as probes or primer pairs and therefore suitable for amplification of sequence fragments using PCR-based methods. Markers describing genetic polymorphisms (between parts of a population) can be detected using established methods from 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, RFLP validation, polynucleotide polymorphism validation by allele-specific hybridization (ASH), detection of amplified variable sequences in plant genomes, detection of 3SR (self-persistent sequence replication), and detection of SSR, SNP, RFLP, or AFLP (amplification fragment length polymorphism). Furthermore, methods for detecting ESTs (expression sequence tags) and SSR markers and RAPD (randomly amplified polymorphic DNA) derived from EST sequences are also known.Depending on the context, the term “marker” as used herein may also mean a specific chromosomal location in the genome of a species where a particular marker (e.g., SNP) may be found.

[0163] The markers also include synthetic oligonucleotides that can be linked to one or more detection molecules, which can be used to generate signals within the scope of the detection reaction or verification method. Synthetic oligonucleotides also include labeled primers. Synthetic oligonucleotides and labeled primers are artificial compounds; they do not exist in nature and cannot be isolated from nature. The preparation of such compounds is further described below.

[0164] A single nucleotide polymorphism (SNP) is a genetic variation in DNA between two samples, where at least one nucleotide differs between the two samples. In most cases, the samples belong to the same species, and the comparison or alignment of the two samples is based on homologous genomic regions. While SNPs can cause allelic variation, not all SNPs need to occur 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, such as a particular gene or its allele variants. Due to genetic linkage, SNPs do not need to be located within the functional genomic elements being selected. Examples of SNPs in the sense of the present invention are shown in Table 1B, and in particular, these are contained within common primer sequences. Detection or identification of SNPs can be performed by PCR using two different forward primers and one common reverse primer. Technical details of such detection or identification can be found in the above sections

[67] -

[70] .

[0165] A "promoter" is a non-coding regulatory DNA sequence, usually upstream of the coding region, that contains the binding site for RNA polymerase and initiates DNA transcription. Promoters further contain other elements (e.g., cis-regulatory elements) that act as regulatory genes for gene expression. A "core promoter or minimal promoter" is a promoter that contains the basic elements required for transcription initiation (e.g., TATA boxes and / or initiators).

[0166] A "pathogen" refers to an organism that, through interaction with a plant, causes disease symptoms in one or more organs of that plant. For example, animals, fungi, bacteria or viruses, or oomycetes are all considered pathogens.

[0167] "Pathogenic infection" is understood as the earliest point at which a pathogen interacts with the host tissue of a plant. In this sense, "invasion" means the occurrence of contact between the pathogen and the host. Once the pathogen settles on the host, for example, when fungal spores settle on the surface of a plant leaf, pathogen detection and signaling mechanisms are initiated in the host cells of the plant. In the case of Cercospora beticola, conidia are formed in humid and warm climates and are transmitted to nearby plants by rain and wind. In new infections, individual leaf spots are often seen on physiologically older outer leaves. These are almost always clearly demarcated from healthy leaf tissue by brown rings. Using a magnifying glass, the brown conidial carriers of the fungus can be observed in the center of the spots (evaluation score 3). The number of these brown spots increases rapidly, initially with sporophytes overlapping to form even smaller dead areas (evaluation score 5). As the disease progresses, it spreads to the inner leaves, eventually causing the outer leaves to wither and fall off first (score 7), followed by virtually all leaves falling off (score 9). The progression of the disease and the severity of the symptoms are heavily influenced by location and weather conditions, which vary from year to year.

[0168] The “organs” of a plant include, for example, leaves, shoots, stems, roots, hypocotyls, buds, meristematic tissue, 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” can also mean a collection of multiple organs, such as a flower or seed, or a part of an organ, such as a cross-section of a plant shoot. The “tissues” of a plant include, for example, callus tissue, storage tissue, meristematic tissue, leaf tissue, shoot tissue, root tissue, plant tumor tissue, or reproductive tissue, as well as the cambium, parenchyma, vascular tissue, chlamydial tissue, and epidermis. However, tissues are not limited to this enumeration. For example, the “cells” of a plant are understood to be, for example, isolated cells with cell walls or aggregates thereof, or protoplasts.

[0169] "Recombination frequency" refers to the occurrence of cross-recombination during meiosis between certain genomic elements, such as single nucleotide polymorphisms, genes, loci, QTLs, or genetic regions. A recombination frequency of 1% corresponds to 1 cM. Recombination frequency can be determined by creating a meiotic map.

[0170] The "resistance gene according to the present invention" is a nucleotide molecule or nucleic acid sequence as described in item [1] above. This gene may be genetically linked to a cosegregated region.

[0171] "Cultivar" means a group of plants within a single plant taxonomic group of the lowest known rank, regardless of whether the conditions for granting breeder's rights are fully met. - Defined by the expression of traits resulting from a given genotype or combination of genotypes, - Distinguish from any other plant population by the expression of at least one of the above characteristics, - It can be considered as a single unit in terms of its suitability for reproduction without changing.

[0172] In connection with the present invention, the term “regulatory sequence” refers to a nucleotide sequence that influences specificity and / or expression intensity, for example, by conferring a predetermined tissue specificity. Such a regulatory sequence may be located not only upstream of the transcription start site of the minimal promoter, but also downstream therein, for example, in a transcribed but untranslated leader sequence, or within an intron.

[0173] The term "resistance" is understood broadly, encompassing a range of protection from delaying disease onset to complete prevention. An example of a significant pathogen is Cercospora beticola. The resistant plant cells or plants of the present invention preferably achieve resistance to Cercospora beticola. Resistance to a pathogen is considered equivalent to resistance to the disease caused by that pathogen; for example, resistance to Cercospora beticola is also resistance to leaf spot disease. For example, increased resistance can be measured by a decrease in fungal biomass on the host plant. For this purpose, fungal DNA can be determined by quantitative PCR compared to plant DNA in the affected plant tissue. An additional approach to measuring resistance is optical evaluation, which is given an evaluation score from 1 (not susceptible) to 9 (very susceptible).

[0174] The term "transgenic plant" refers to a plant in which at least one polynucleotide is incorporated into its genome. Therefore, this may be a heterogeneous polynucleotide. The polynucleotide is preferably stably incorporated, i.e., the incorporated polynucleotide can be stably retained, expressed, and even stably passed on to offspring in the plant. Stable introduction of a polynucleotide into the plant genome includes integration into the genome of a previous parental generation of plant, and the polynucleotide can be stably passed on further. The term "heterogeneous" means that the introduced polynucleotide originates from a cell or organism with a different genetic background, either homogeneous or heterogeneous, or is homologous to, for example, a prokaryotic or eukaryotic host cell but is different from a naturally occurring corresponding polynucleotide due to being placed in a different genetic environment. Heterogeneous polynucleotides may be present in addition to the corresponding endogenous gene.

[0175] "Industrial sugar raw materials" refer to plant materials that can be supplied to sugar production facilities specifically designed for extracting sugar from sugar beets. Such raw materials are typically the taproots of harvested sugar beets. To ensure compatibility with the extraction process, the taproots must have sufficient mass, volume, and conical shape so that they can be mechanically cut into small pieces (beet strips). These beet strips must have a high surface area for sugar extraction and low sodium, potassium, and nitrogen content to enable efficient extraction. The remaining beet pulp after extraction is pressed, dried, and used as animal feed.

[0176] "Saccharose concentration" is expressed as a percentage of the fresh weight of the root.

[0177] "Monoembryonic" means that the seed develops into exactly one plant, while polygermous or multiembryonic seeds (also called "seed bulbs") develop into several plants.

[0178] "Bolting" refers to the natural process by which sugar beets develop flowering stalks (or stems) as they attempt to produce seeds and reproduce. In sugar beets, vernalization, or cold stress that can occur, for example during overwintering, induces bolting. However, commercially cultivated sugar beets are harvested before bolting because the bolting process and subsequent fruiting reduce the saccharose content within the beet.

[0179] "Genetic transfer" means that a nucleotide sequence has been introduced into the genome of a plant, and this nucleotide sequence originates from a plant that does not belong to the same species or subspecies. This could mean, for example, that a nucleotide sequence from a plant of the subspecies Beta vulgaris maritima has been introduced into a plant of the subspecies Beta vulgaris vulgaris.

[0180] The design and embodiments of the present invention will be described by reference to the attached arrangement and drawings. [Brief explanation of the drawing]

[0181] [Figure 1] This diagram shows the protein sequence alignment between a resistance protein (a protein that confers circospora resistance in plants) and a susceptibility protein (a protein that does not confer circospora resistance in plants). Polymorphisms are highlighted in gray. [Figure 2] These are gene maps (left) and physical maps (right) of genomic regions containing resistance genes, showing the locations of some of the most important markers. [Figure 3] This is a vector map of the vector pZFN-nptII, which includes the LRR region. [Figure 4] This figure shows a statistical box plot evaluation of data generated 8 days after infection in transgenic validation of resistance genes. [Figure 5] This figure shows a statistical box plot evaluation of data generated 11 days after infection in transgenic validation of resistance genes. [Figure 6] This figure shows a statistical box plot evaluation of data generated 8 days after infection in transgenic validation of resistance genes. [Figure 7] This figure shows a statistical box plot evaluation of data generated 15 days after infection in transgenic validation of resistance genes.

[0182] Detailed description of the invention This invention relates to nucleic acid molecules that can confer resistance to Circospora in plants, particularly Beta vulgaris subsp. vulgaris, by expressing polypeptides encoded by nucleic acid molecules. According to a preferred embodiment of the invention, the pathogen is the fungus Circospora beticola, one of the most important and destructive leaf pathogens, particularly in sugar beets, beetroot, and chard, which can cause crop losses of more than 40%. This fungus reacts with oxygen in the presence of light to produce the secondary metabolite cercosporin, which leads to the formation of reactive oxygen species (ROS). ROS cause significant cellular damage to the leaf tissue of affected plants, which is visible in the form of necrosis.

[0183] This invention is based on the detailed genetic mapping, identification, isolation, and characterization of genes or loci derived from Beta vulgaris subsp. maritima donors whose presence correlates with or contributes to the plant's resistance to circospora leaf spot disease in plants, particularly Beta vulgaris subsp. vulgaris. The initial material was a population of Beta vulgaris subsp. maritima generated from 37 Beta vulgaris subsp. maritima accessions from different origins. Seeds of the Beta vulgaris subsp. vulgaris plant (beet cultivar 6626) possessing resistance according to the present invention were deposited prior to the filing of this application with the National Collection of Industrial Food and Marine Bacteria (NCIMB) in Aberdeen, UK, under access number NCIMB 43646. In particular, plants of the genus Beta vulgaris, plants of the Beta vulgaris species, or plants of the Beta vulgaris subsp. vulgaris subspecies, or beet plants containing the resistance gene according to the present invention may be derived from deposited seeds.

[0184] The nucleotide and amino acid coding sequences of nucleic acid molecules according to the present invention are characterized by numerous polymorphisms, which distinguish the NPS-LRR gene identified according to the present invention from "sensitive" variants of the gene, i.e., variants of the gene that do not confer resistance to circospora. Examples of polymorphisms are shown in Figure 1.

[0185] The nucleic acid molecule according to the present invention may be an isolated nucleic acid molecule. The nucleic acid molecule is preferably DNA, and more preferably cDNA (coding DNA). The polypeptide encoded by the nucleic acid molecule according to the present invention preferably confers resistance to the pathogen Circospora beticola, which causes the plant disease Circospora leaf spot. Furthermore, the polypeptide encoded by the nucleic acid molecule according to the present invention confers resistance to this pathogen, particularly in plants of the genus Beta vulgaris. The plants are preferably plants of the species Beta vulgaris, and more preferably plants of the subspecies Beta vulgaris vulgaris, which include, for example, cultivated varieties of sugar beet, beetroot, fodder beet, chard, and Swiss chard.

[0186] In one embodiment of the present invention, the nucleic acid molecule according to the present invention comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence according to SEQ ID NO: 3 and / or a coding DNA sequence according to SEQ ID NO: 2. Furthermore, the present invention provides a nucleotide sequence comprising DNA sequences according to SEQ ID NO: 1 and SEQ ID NO: 53.

[0187] The genes identified according to this invention are NBS-LRR type resistance genes / proteins characterized by specific structural motifs. The overall structure of such resistance proteins in plants has already been thoroughly investigated (Martin et al., Annual Review Plant Biology 54 (2003), 23-61). However, the principle of structural embodiment of the so-called LRR domain, which is applied as a potential detection domain for most unknown pathogenic effectors, is unpredictable, and the functional background of resistance genes, i.e., the gene structure, is generally largely unknown. Identification of circospora resistance-constituting genes or proteins based solely on known structural motifs is consequently impossible. Furthermore, this sequence region has been found to be a high-frequency repeat region containing tandem repeats with particularly high sequence homology, which makes the development of diagnostic markers and the aggregation of sequence data particularly difficult.

[0188] By establishing a population of over 4000 differentiated offspring and developing a special recombination screen, the target region was narrowed and further isolated through analysis of informative recombinants (genotype and phenotype) in a series of resistance tests. This gene mapping, along with the creation of physical maps using WHG sequencing ("whole-genome sequencing"), comparative BAC (Bac-by-Bac) sequencing, and bioinformatics analysis, identified three recombinant genotypes that confirmed the resistance gene (one with one recombinant in a neighboring gene, and the other with two recombinants in neighboring genes). Considering specific requirements, the inventors placed high-frequency repeat structures, including tandem repeats with particularly high sequence homology, in the target region, which made marker development and, consequently, the identification of informative recombinants extremely difficult. The following steps were particularly critical to the location of the resistance gene's genetic structure: - Development of markers s4p0264s01, s4p2271s01, sxh0678s01, s4p4293s01, s4p4295s01, and s4p4301s01 (see Table 1B). - Detailed mapping combined with intensive phenotyping. In greenhouse experiments, 90 to 180 offspring were used per plant, and phenotypic analysis was performed using intensive statistical methods (e.g., t-tests, power analysis, etc.). - Identification and sequencing of BAC clones from a BAC pool of resistance genotypes. - Sequence evaluation, as well as the comparison of sequences and proteins between RR (i.e., resistant) and ss (i.e., susceptible) genotypes; in this regard, clear aggregation of RR and ss sequence data was not always possible due to the complexity of the sequences.

[0189] [Table 2-1] [Table 2-2]

[0190] The compounds presented in Table 1B can be used as molecular markers according to the present invention. All markers are suitable for detecting genetic material containing the resistance gene according to the present invention (particularly by identifying genetically linked polymorphisms such as single nucleotide polymorphisms) and for tracking the inheritance of genetic material from the origin plant to its offspring by marker-assisted selection (MAS). In this regard, the markers disclosed herein may be very useful for transmitting the resistance gene according to the present invention from one subspecies to another. The markers disclosed herein can be further used for gene mapping and tracking recombination as a result of meiosis. The resistance gene appears to be located near or between the physical locations 57219956 bp and 57243521 bp. The markers disclosed herein located very close to this region are very suitable for detecting the resistance gene. The markers disclosed herein located further away from the resistance gene still show strong genetic linkage with the resistance gene but can be further used for detecting rare recombination events and gene exchanges in the adjacent region of the resistance gene. This may be useful, for example, for backcross approaches and for transmitting the resistance gene to different genetic backgrounds. The markers disclosed herein, particularly those located further away from the resistance gene, are especially useful when the resistance gene is transmitted to offspring as part of a co-segregated region. Resistance genes containing co-segregated regions may also be transmitted from one subspecies to another. Successful transmission can be confirmed by the markers disclosed herein, for example, by detecting the resistance gene or the co-segregated region. The markers disclosed herein can also be used in backcross programs involving several generations to reduce the co-segregated region while retaining the resistance gene according to the present invention in the offspring. Further markers for detecting the resistance gene according to the present invention can be inferred from the data disclosed herein. In particular, markers located in genomic intervals adjacent to the markers s4p0421s01 and s4p1395s01, or adjacent to the marker pair shown in item

[48] above, can be inferred. The inference of further markers is facilitated by the genomic material contained in seeds deposited at the NCIMB in Aberdeen, UK, with access number NCIMB 43646.This genomic material allows for, for example, alignment of genetic material encoding resistance according to the present invention with genetic material lacking the resistance gene according to the present invention. The resistance-lacking genetic material may be homologous to the resistance-encoding genetic material according to the present invention, or be at the same physical location (compare Table 1B). These alignments can be used to identify polymorphisms, such as single nucleotide polymorphisms. Based on the polymorphisms, additional markers can be created for the detection of identified polymorphisms, particularly those genetically linked to the resistance gene according to the present invention. Alignment and the detection of polymorphisms based on alignments typically involve the use of computers and the storage of data on computer-readable media. It is also proposed to convert signals generated by molecular markers during the analysis of genetic material into electronically transmittable and / or electronically storable data that can be processed using a computer. Such computer-based processes may include determining or calculating the estimated breeding value of a plant. The estimated breeding value may encompass the level of resistance or tolerance to Circospora. Further information regarding the estimation of breeding values ​​can be found in U.S. Patent No. 8,321,147.

[0191] Analysis revealed that the LRR gene has moderate protein homology to the tomato-derived Cf-2 resistance protein (UNIPROT|Q41397_SOLPI P. Cf-2.1) (sequence identity 322 / 830 = 38%). In fact, the identified Circospora resistance-conferring protein is the best sugar beet protein homolog to the Cf-2 tomato resistance protein. The tomato-derived Cf-2 resistance protein confers resistance to Cladosporium fulvum, a type of black mold (U.S. Patent No. 6,287,865), through interaction with the non-pathogenic protein Avr2 derived from C. fulvum. This leads to the activation of plant 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). The sequence homology between the Cf-2 gene and the identified LRR gene suggests that a similar defense mechanism underlying circospora resistance also occurs in sugar beets, but this does not restrict us to a single theory. Furthermore, moderate sequence homology should not rule out different mechanisms.

[0192] Furthermore, substitutions, deletions, insertions, additions, and / or any other changes that actually alter the nucleotide sequence, either alone or in combination, may be introduced into the nucleotide sequence according to the present invention, but the modified nucleotide sequence may perform the same function as the original sequence. In this case, we are dealing with the coding of an amino acid sequence that confers resistance to Circospora leaf spot disease. Thus, in further embodiments, the present invention includes a derivative of the polypeptide encoded by the nucleotide sequence according to the present invention, or a nucleotide sequence encoding a polypeptide comprising the amino acid sequence according to the present invention. An derived amino acid sequence having at least one substitution, deletion, insertion, or addition of one or more amino acids, in which the functionality of the encoded polypeptide / protein is preserved, is a derivative of the polypeptide. Thus, substitutions, deletions, insertions, additions, and / or any other changes that actually alter the nucleotide sequence, either alone or in combination, but perform the same function as the original sequence, can be introduced into the nucleotide sequence using conventional methods known in the prior art, for example, by site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, genome editing, etc.

[0193] Substituting an amino acid with a different amino acid having the same, equivalent, or similar chemical / physical properties is referred to as a "conservative substitution" or "semi-conservative substitution." Examples of the physical / chemical properties of amino acids include, for example, hydrophobia or electric charge. Which amino acid substitutions are conservative or semi-conservative is known to those skilled in the art. Furthermore, with general expertise, those skilled in the art can recognize, identify, and detect which amino acid deletions and additions are harmless to the functionality of the resistance protein, and at what positions these are possible. In the case of the NBS-LRR protein, it is known to those skilled in the art that any modification of the amino acid sequence (substitution, deletion, insertion, or addition of one or more amino acids) must preserve the functionality of the conserved domains in particular, and therefore only a limited number of prior modifications are possible in these domains.

[0194] Accordingly, the present invention includes functional fragments of nucleotide sequences according to the present invention. The term “fragment” thereby includes genes having nucleotide sequences that are sufficiently similar to the nucleotide sequences described above. The term “sufficiently similar” means that the first nucleotide sequence or amino acid sequence has a sufficient or minimal number of identical or equivalent nucleotides or amino acid groups to the second nucleotide sequence or second amino acid sequence.

[0195] With respect to amino acid sequences, they have a common structural domain and / or common functional activity after modification by the method described above. A nucleotide sequence or amino acid sequence having at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least approximately 100% identity with the nucleotide sequence or amino acid sequence of the present invention is defined herein as sufficiently similar. The range of 90% to 100% is also explicitly included. With respect to functional fragments, sufficient similarity is established if the nucleotide sequence or amino acid sequence generally has the same characteristics as the nucleotide sequence or amino acid sequence of the present invention described above. Those nucleotide sequences encoding derivative or derived amino acid sequences are generated directly or indirectly (e.g., by amplification or replication steps) from the initial nucleotide sequence corresponding to the nucleotide sequence of the present invention, either over its entire length or at least partially.

[0196] Therefore, the present invention includes a nucleotide sequence that can hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the nucleotide sequence or amino acid sequence according to the present invention.

[0197] In further embodiments, the nucleic acid molecule according to the present invention is characterized by encoding a polypeptide that, after expression in a plant, already confers a dominant resistance effect to a pathogen, preferably Cercospora beticola, or can confer a dominant resistance effect to Cercospora. In preferred embodiments, the nucleic acid molecule or polypeptide confers a resistance effect of at least 1 evaluation score, preferably at least 2 evaluation scores, and particularly preferably 3-4 evaluation scores. No gene is known from the prior art that already encodes a polypeptide that already confers such a remarkably high resistance to Cercospora in a plant, or can confer such a remarkably high resistance. As already mentioned above, in varieties that have been available on the market until now, Cercospora resistance is transmitted via many resistance genes that have little effect, and the disadvantages of such varieties are that their development is very slow and costly due to the complex transmission, and that such varieties have significantly lower crop yields compared to normal varieties when not invasive. In particular, this can be attributed to epigenetic interactions between several resistance genes and genes involved in sugar production, resulting in reduced plant fitness in the absence of the pathogen.

[0198] Thus, the inventors have for the first time provided a circospora resistance gene that can be used to significantly simplify breeding. By incorporating this gene into superior lines, it has become possible to develop very high-yielding varieties with high circospora resistance very quickly. Therefore, within the framework of the present invention, sugar beet plants, chard plants, red beet or beetroot plants, and fodder beet plants that are resistant to circospora beticola according to the present invention and are therefore included in the present invention are provided for the first time. Since all the listed plants are cultivated plants, crops or plants that are suitable for agricultural cultivation and are resistant according to the present invention are part of the present invention. In particular, such crops are part of the present invention and include underground storage organs that can be used as food, raw materials, or industrial sources of sugar, and crops that are resistant according to the present invention are further embodiments of the present invention. The storage organs may be, for example, sugar-containing beets of sugar beets, consumable beets of red beets, or feedable beets of fodder beets. The underground storage organ can account for more than 50% of the total mass of a fully grown plant, and more than 70% in the case of sugar beets. Furthermore, the seeds or seedling material of these plants are also part of the present invention. The seeds or seedling material can be technically processed as further described below. Plants of the genera Spinach, Glycine, Carrot, and Angelica containing the resistance gene according to the present invention are also part of the present invention. In particular, this includes plants of the species Spinacia oleracea and its varieties containing the resistance gene according to the present invention. The storage organ may be the taproot, especially the beet body.

[0199] The resistance gene according to the present invention may be under the control of the promoter by SEQ ID NO: 7, or may be operably ligated to it. Alternatively, the resistance gene may be under the control of a nucleic acid molecule containing the promoter by SEQ ID NO: 7, or may be operably ligated to it. Sequences having 90%, 95%, or 99% sequence identity with SEQ ID NO: 7 across the entire length of the sequence are included in this regard.

[0200] In this regard, the present invention also includes nucleic acids encoding proteins according to Sequence ID No. 3, and in certain embodiments, nucleic acids according to Sequence ID No. 1 are excluded.

[0201] Furthermore, the present invention relates to recombinant and / or heterologous DNA molecules comprising a sequence of nucleic acid molecules according to the present invention. The DNA molecule preferably further comprises a regulatory sequence, which may be operably linked to or under the influence of this regulatory sequence. The regulatory sequence is preferably a promoter sequence and / or other sequences of transcriptional or translational regulatory 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 that can confer or regulate expression as a result of pathogenic infection. The promoter can preferably specifically control the expression of the DNA sequence in the leaves of a plant. The regulatory sequence may be heterologous to the expression sequence. Such an approach has the advantage that a person skilled in the art can better control the expression rate of the expression sequence, the tissue in which expression occurs, and the timing of expression, in that they can select the most suitable regulatory sequence for each use case. The heterologous DNA sequence preferably comprises a nucleotide sequence encoding components of plant pathogen defense (e.g., resistance genes (R genes), or enzymes involved in signal transduction, such as kinases or phosphatases, and G proteins, or genes encoding pathogenicity effectors (so-called non-pathogenic genes (avr))). The heterologous DNA sequence may be one of the DNA sequences according to the present invention. The heterologous DNA sequence may additionally encode further components of plant pathogen defense. Therefore, the heterologous DNA sequence can be designed so that polycistronic mRNA is produced after its transcription.

[0202] The present invention also relates to polypeptides that can be encoded by nucleic acid molecules according to the present invention, as well as functionally and / or immunologically active fragments thereof, and antibodies that specifically bind to the polypeptide or its fragment. The polypeptide particularly preferably has the amino acid sequence according to SEQ ID NO: 3. The recombination of proteins, polypeptides, and fragments is well known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 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 the proteins according to the present invention can be prepared according to methods known to those skilled in the art (E. Harlow et al., editor, Antibodies: A Laboratory Manual (1988)). Monoclonal antibodies, as well as Fab and F(ab')2 fragments useful for protein detection, can be prepared by various conventional methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118, New York: Academic Press (1983)).The antibody can then be used to screen an expression cDNA library to identify identical, homologous, or heterologous genes by 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 it can be used for Western blot analysis. In particular, the present invention relates to an antibody that selectively detects polypeptides encoded by the circospora resistance-constitutive allele according to the present invention and does not essentially detect polypeptides encoded by the corresponding susceptibility allele, i.e., the antibody detects twice, preferably five times, more preferably ten times less polypeptides encoded by the corresponding susceptibility allele than polypeptides encoded by the circospora resistance-constitutive allele according to the present invention.

[0203] In preferred embodiments, the antibody according to the present invention is characterized by being a synthetic polypeptide that does not exist in nature.

[0204] Furthermore, the antibodies according to the present invention can be used, for example, in immunohistochemical methods and can be conjugated with a fluorescent dye to induce antibody coloration. The fluorescent dye may be fluorochrome. The antibodies according to the present invention can also exist conjugated with other signaling molecules. These include, for example, biotin, radioisotopes, reporter enzymes such as alkaline phosphatase, or oligonucleotides.

[0205] An additional subject of the present invention is a vector or expression cassette comprising nucleic acid molecules or recombinant DNA molecules according to the present invention, and negative and / or positive selection markers, optionally under the control of regulatory elements in plants, particularly functional regulatory elements. Thus, the vector backbone is heterogeneous to the nucleic acid molecules according to the present invention; i.e., such vectors do not exist in nature and cannot be isolated from nature. The vectors are plasmids, cosmids, phages, or expression vectors, transformation vectors, shuttle vectors, or cloning vectors, and can be double-stranded or single-stranded, linear or circular, or can transform prokaryotes or eukaryotes by integration into the genome or extrachromosomally. The nucleic acid molecules or DNA molecules according to the present invention in the expression vector or expression cassette are preferably operably ligated to one or more regulatory sequences that enable transcription, optionally expression, in prokaryotic or eukaryotic cells (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). These regulatory sequences are preferably promoters or terminators, particularly transcription start sites, ribosome binding sites, RNA processing signals, transcription termination sites, and / or polyadenylation signals. For example, nucleic acid molecules are under the control of preferred promoters and / or terminators. Preferred promoters may be constitutive promoters (e.g., the 35S promoter derived from cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812); pathogenically inducible promoters are particularly preferred (e.g., the PR1 promoter derived from parsley (Rushton et al., EMBO J. 15 (1996), 5,690-5,700))).Particularly suitable pathogenically inducible promoters are synthetic or chimeric promoters that do not exist in nature, are composed of multiple elements, include a minimal promoter, and have at least one cis-regulatory element upstream of the minimal promoter, the at least one cis-regulatory element functioning as a binding site for a specific transcription factor. The chimeric promoter is designed according to desired requirements and is induced or repressed by different factors. Examples of such promoters can be found in International Publications 00 / 29592, 2007 / 147395, and 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 native promoters and native terminators, their DNA sequences reproduced in SEQ ID NOs. 7 and 8. Vectors or expression cassettes often include conventional indicator / reporter genes or resistance genes to detect the transfer of the desired vector or DNA / nucleic acid molecule and select individuals containing them, as direct detection by gene expression is usually quite difficult. Here, since the nucleic acid molecule according to the present invention itself encodes a polypeptide that confers resistance to Circospora leaf spot disease, it is not essential to provide an additional resistance gene for expression in plant cells, but it is recommended to enable rapid selection.

[0206] Examples of indicator / reporter genes are, for example, luciferase genes and genes encoding green fluorescent protein (GFP). These also allow for testing of the activity and / or regulation of the gene promoter. Examples of resistance genes, particularly resistance genes for plant transformation, are neomycin phosphotransferase genes, hygromycin phosphotransferase genes, or genes encoding phosphinotrysin acetyltransferase. Additional positive selection markers may be enzymes that provide a selective advantage to transformed plants over non-transformed 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 preferred embodiments, the vector is a plant vector. Furthermore, the expression cassette may be integrated into the plant genome.

[0207] In further embodiments, the present invention relates to cells comprising vectors, recombinant DNA molecules, and / or nucleic acid molecules according to the present invention. Cells in the sense of the present invention may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., plant cells or yeast cells). The cells are preferably Agrobacterium such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, Escherichia coli cells, or plant cells, and the plant cells are particularly preferably cells of plants of the genus Beta vulgaris, species Beta vulgaris, or subspecies Beta vulgaris vulgaris. The cells may exist as a culture. The present invention also consequently encompasses cell cultures containing such cells. The cell culture is preferably a pure culture or isolate that does not contain other types of cells.

[0208] Numerous methods, such as conjugation or electroporation, for introducing nucleic acid molecules, recombinant DNA molecules, and / or vectors or expression cassettes according to the present invention into Agrobacterium, as well as various transformation methods (bioristic transformation, Agrobacterium-mediated transformation, etc.) for introducing nucleic acid molecules, DNA molecules, and / or vectors according to the present invention into plant cells, are known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0209] Furthermore, the present invention relates to a circospora-resistant plant, preferably a plant of the Beta vulgaris subsp. vulgaris species or a part thereof, which preferably contains the nucleic acid molecule according to the present invention that confers circospora resistance. The circospora-resistant plant may contain the nucleic acid molecule according to the present invention as an introduced gene or an endogenous gene. Within the scope of the present invention, a plant of the Beta vulgaris subsp. vulgaris subspecies containing the nucleic acid molecule according to the present invention has been created for the first time. Hereinafter, the present invention also includes a plant of the Beta vulgaris subsp. vulgaris subspecies containing the nucleic acid molecule according to the present invention as an endogenous gene.

[0210] This means that a part can be a cell, tissue, organ, or a combination of multiple cells, tissues, or organs. A combination of multiple organs is, for example, a flower or a seed. The circospora-resistant plants of the present invention preferably exhibit higher resistance to circospora, particularly circospora beticola, than the corresponding plants (control plants) that do not contain the nucleic acid molecules according to the present invention. The control plants ideally have the same genotype as the transgenic plants and are grown under the same conditions, but do not contain the resistance-constituting nucleic acid molecules. For example, the level of resistance to circospora beticola can be qualitatively established by determining an evaluation score in plants of the genus Chamaecyparis. Higher resistance manifests as an improvement in resistance of at least 1 evaluation score, at least 2 evaluation scores, preferably at least 3 evaluation scores or more.

[0211] Plant cells or plants or parts thereof containing the nucleic acid molecule according to the present invention, particularly plants of the genus Cercospora, preferably exhibit higher resistance to pathogens, particularly Cercospora beticola, than corresponding plant cells or plants or parts thereof that do not contain the nucleic acid molecule according to the present invention or that contain a susceptible allele variant of the nucleic acid molecule. For example, the level of resistance to Cercospora beticola can be qualitatively established by determining an evaluation score in plants of the genus Cercospora. Higher resistance manifests as an improvement in resistance of at least 1 evaluation score, at least 2 evaluation scores, preferably at least 3 evaluation scores or more.

[0212] In the case of transgenic plant cells or plants or parts thereof, this includes a nucleic acid molecule or DNA molecule according to the present invention as a transgene, or a vector or expression cassette according to the present invention. Such transgenic plant cells or plants or parts thereof are preferably stably transformed with, for example, a nucleic acid molecule, DNA molecule, or vector or expression cassette according to the present invention. In a preferred embodiment, the nucleic acid molecule is operably linked to one or more regulatory sequences that enable transcription, optionally expression, in the plant cell. The overall structure consisting of the nucleic acid molecule and regulatory sequences according to the present invention in this case constitutes a transgene. Such regulatory sequences are, for example, promoters or terminators. Numerous functional promoters and terminators applicable in plants are known to those skilled in the art.

[0213] The present invention also includes the vacuole of a cell according to the present invention and the contents stored therein.

[0214] Furthermore, the present invention also relates to cells, preferably plant cells, particularly preferably cells of Beta vulgaris, and especially preferably cell extracts from one of the following crops: sugar beet, chard, or beetroot. It is not possible to regenerate a plant from a cell extract.

[0215] Similarly, a plant genome containing nucleic acids according to the present invention is included in the present invention.

[0216] This allows the sugar concentration from cell extracts to be increased compared to cells that are not from the present invention but belong to the same species or crop. This is especially true under the conditions of circospora invasion.

[0217] The use of cell extracts for the production of sugars (saccharose) or juice, preferably beetroot juice, is also included in the present invention.

[0218] Similarly, the sugars contained in the cells and vacuoles of the present invention, particularly saccharose, are included in the present invention.

[0219] An additional aspect of the present invention is a seed stock comprising seeds containing nucleic acids according to the present invention. The nucleic acids according to the present invention may be present genetically or endogenously. Seed stocks and seeds may be technically processed. For this reason, the present invention also includes technically processed seed stocks and technically processed seeds. Various embodiments of technically processed seed stocks are described in detail below, and the term seed stock also includes seeds. Technically processed seed stocks may exist in a polished form. The outermost layer of the seed is removed thereby, and the seed becomes more rounded. This is helpful for sowing, and the optimally uniform shape leads to a uniform distribution of seed stock grains. Technically processed seed stocks further encompass pelletized seed stocks. This allows the seed stock to be embedded in a pelletized mass that protects the seed stock contained within and gives it a larger mass, and pelletized seed stocks exhibit greater resistance to windflow, making them less likely to be blown away by the wind, and at the same time allowing for more precise positioning during sowing. In a preferred embodiment of the present invention, all pelletized seed stock grains in a designated batch or unit for sale have essentially the same shape and mass. A deviation of 5% in diameter and mass is possible. However, the deviation preferably does not exceed 1%. As one of the main components, the pellet mass may contain mineral compounds such as clay, bentonite, kaolin, humus and / or peat. Adhesives such as polyacrylamide can be added. Possible additional components are listed in U.S. Patent No. 4,067,141. Furthermore, the pellet mass may contain additional chemicals that have a practically beneficial effect on cultivation. These chemicals may be substances that are counted here as fertilizers. These include compounds rich in one or more of the following elements: nitrogen, phosphorus and potassium (macromolecules). Thus, fertilizer components may include, for example, nitrate nitrogen, ammonium nitrogen, magnesium nitrate, calcium ammonium nitrate, monoammonium phosphate, monopotassium phosphate and potassium nitrate. Furthermore, the pellet mass may contain fungicides, insecticides and / or feeding inhibitors.The fungicide may be thyram and / or himexazole and / or other fungicides. The insecticide may be a neonicotinoid substance. The neonicotinoid substance is preferably imidacloprid (ATC code: QP53AX17) and / or clothianidin (CAS number 210880-92-5). Furthermore, the insecticide may be cyfluthrin (CAS number 68359-37-5), β-cyfluthrin, or tefluthrin. It is worth noting that the compounds contained in the powder or pellet mass are taken up by the plant, exerting a systemic effect and thereby providing favorable protection for the entire plant. Therefore, plants obtained from pelletized seeds containing one or more insecticides exhibit better performance under biological stress conditions than naturally occurring plants. In this regard, the present invention also encompasses mixtures of pellet mass and seeds according to the present invention. Furthermore, the present invention provides a method for producing pelletized seeds according to the present invention, comprising the following steps: a) A step of preparing plant seeds containing nucleic acids according to the present invention, b) The process of embedding plant seeds into a pellet mass, and c) A process of air-drying the pellet mass or drying the pellet mass. The method also includes a method in which the plant seeds may optionally be primed or pre-germinated plant seeds, or the plant seeds may be primed during step b). Optionally, the method may include an additional step d): d) The process of packaging the embedded plant seeds obtained in step c) into a packing. This can be the end of the packing process. Examples of suitable packing are shown in other parts of this specification. The plant seeds may be, for example, seeds of plants of the genus Bhutan described herein, including sugar beet or red beet.

[0220] The seed pellets may contain beneficial bacteria. Preferably, the bacteria are in an active state and can grow. Symbiotic bacteria can have various beneficial effects on seed germination, including increased vigor, increased germination rate, and better resistance to stress. According to a preferred embodiment, a microorganism of the genus Pseudomonas is incorporated into the seed pellets. Additional strains of different bacteria may be added. A possible method for improving the survival rate of the microorganisms is a) suspending one or more precultures of microbial cells in a polymer substance solution, b) immobilizing the microbial cells by dropping the solution of step a) into a polyvalent ion solution to obtain polymer particles, c) culturing the encapsulated microorganisms in the polymer particles in a liquid medium for at least 12 hours, preferably at least 24 hours, until an increase in cell density of at least 2 to 10 log is obtained, d) recovering the polymer particles, and e) drying the polymer particles is included. Pelleted seeds containing the resistance gene according to the present invention and further containing the microorganisms obtained by the method disclosed in this section are part of the present invention. Further, the microorganisms may be encapsulated and / or embedded in an extracellular matrix containing naphthazarin.

[0221] In this regard, technically treated seed stocks also include powdered seed stocks. The present invention can be applied to any form of powdered seed stock. Therefore, dry powdering, wet powdering, and suspension powdering are also included. Thus, the powdering may contain at least one dye (colorant), allowing for rapid distinction between powdered and unpollinated seed stocks, and further ensuring good visibility in the environment after sowing. The powdering may contain pesticides as described in relation to pilling masses. Therefore, the present invention includes such powdered seed stocks in which the powdering contains at least one feeding inhibitor and / or at least one fungicide, such as an insecticide. Optionally, so-called electronic dressing (dressing by applying electrical energy) may be applied. Although electronic dressing is not powdering in the strict sense of the word, it is very suitable for destroying plant pathogens attached to seeds or seed stocks before sowing the seeds or seed stocks. When more seeds or seed stocks are available than are needed for cultivation, it is also beneficial to feed animals seeds or seed stocks that have been treated only with electrocoating (without the use of pesticides).

[0222] An additional form of technically treated seed stock is a coated seed stock. So-called coatings are discussed in relation to this, as are coated seed stocks. The difference from pelletized seed stocks is that the seeds retain their original shape, and this method is particularly economical. This method is described, for example, in European Patent Application Publication No. 0334258. Additional forms of technically treated seed stocks are sprouting or priming seed stocks. Sprouting seed stocks are pre-treated by pre-germination, and priming seed stocks are pre-treated by priming ("germination"). Pre-germination and priming seed stocks have the advantage of a shorter emergence time. At the same time, the timing of sowing and emergence is more strongly synchronized. This allows for better agricultural technical treatment during cultivation, especially at harvest, and further increases yield. In pre-germination, the seed stock is germinated until the radicle emerges from the seed stock shell, and then the process is stopped. In priming, the process is stopped before the radicle emerges from the seed stock shell. Compared to pre-germinated seed stock, priming-treated seed stock is unaffected by the stress of re-drying and, after such re-drying, has a longer shelf life compared to pre-germinated seed stock, for which re-drying is generally not recommended. In this regard, technically pre-treated seed stock includes primed and re-dried seed stock. The pre-germination process is described in U.S. Patent No. 4,905,411. Various embodiments of priming are described in European Patent Application Publication No. 0686340. In addition, it is also possible to pelletize and prime seed stock simultaneously in a single process. This method is described in European Patent No. 2002702. Furthermore, pelletized primed seed stock is also encompassed in the present invention.

[0223] Technically treated seed stocks may additionally possess one or more of the herbicide resistances described above. This allows for further improved agricultural cultivation by enabling the sowing of technically treated seed stocks in fields that have previously been treated with herbicides and thus completely weeded.

[0224] In addition, the present invention also encompasses a seed stock according to the present invention or a mixture comprising seeds according to the present invention and the powdered mass as defined above. The powdered mass is preferably embodied as the pellet mass as defined above.

[0225] In the storage of seed stock according to the present invention, storage conditions are preferably selected so as not to adversely affect the stability or shelf life of the seed stock. Here, in particular, fluctuations in humidity can have an undesirable effect. A method of storing seed stock in a bag or container that is both water-repellent and breathable is part of the present invention. Such a bag or container can be designed as a carton or packing. Such a carton or packing may optionally have an inner vapor barrier. If the carton or packing is designed as a double carton, its stability is increased. Containers, bags, cartons or packing containing seed stock or technically treated seed stock according to the present invention are also part of the present invention. Storing seed stock or technically treated seed stock according to the present invention in such bags, containers, boxes, packings or cartons is also part of the present invention.

[0226] The present invention also includes varieties containing the resistance gene according to the present invention. Furthermore, it includes plants, seeds, and seed stocks of such varieties. Seeds and seed stocks of such varieties can be subjected to the technical processing described herein (e.g., pelletizing). Sugar beet varieties suitable for introducing the resistance gene include, for example, BTS 7300 N, BTS 2045, BTS 3750, DAPHNA, KORTESSA KWS, or SABATINA KWS. Sugar beet plants of the specified varieties are also examples of hybrid sugar beet plants. Red beet varieties suitable for introducing the resistance gene include, for example, Jolie, Scarlett (PV-9503), or Diaz, with Jolie and Diaz also being examples of hybrid red beet plants. Swiss chard varieties suitable for introducing the resistance gene include, for example, Fluence, Ion, or Tesla / PV-9022. Suitable varieties of Spinacia oleracea (spinach) for introducing resistance genes include, for example, PV-9210, PV-1194, or La Paz / PV-1237. Hybrid plants utilize the effect of hybrid vigor.

[0227] In one embodiment, the plant according to the present invention is a hybrid plant or a doubling haploid plant. Hybrid plants and doubling haploid plants do not exist in nature and cannot be isolated from nature. In a further embodiment of the plant according to the present invention, the nucleic acid molecule according to the present 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 present invention also encompasses hybrid seeds and doubling haploid seeds containing the nucleic acid or polypeptide according to the present invention.

[0228] Further embodiments of the present invention include plants, preferably of the Beta vulgaris species, characterized by further increased resistance to Circospora in these plants. For example, this can be achieved by "gene stacking," i.e., by increasing resistance using this dose-effect. To this end, plants according to the present invention containing a Circospora resistance-constitutive allele are overtransformed with this resistance allele to increase the transcription level of the gene in the plant. Alternative approaches include gene editing / site-directed mutagenesis or tilling-mediated modification of the native promoter of the resistance-constitutive allele to increase its expression rate, or modification of the allele of the resistance-constitutive LRR gene itself to increase its activity or stability. Methods for increasing activity by such modification of the resistance gene are described, for example, in International Publication No. 2006 / 128444 and can be carried out by methods known to those skilled in the art. An additional approach is the fusion of the nucleic acid molecule according to the present invention with a heterologous promoter that exhibits higher activity compared to the native promoter, particularly after Circospora infection.

[0229] An additional embodiment of the present invention relates to sugar beet plants or a portion thereof, or pelletized seeds of such plants, in which bolting does not occur between 10, 11, 12, 13, 14, or 15 months from germination, and the development of the beet body is completed during this period, making them harvestable before bolting. Suitable varieties for producing sugar beet plants according to this section by introducing resistance according to the present invention are, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0230] In one embodiment of the present invention, a sugar beet plant or a portion thereof, or pelletized seeds of such plant, has a genome that enables the development of a beet body having a mass reaching at least 50%, 60%, 70%, 80%, or even 90% of the total mass of a fully grown plant. Suitable varieties for producing sugar beet plants according to this section by introducing resistance according to the present invention include, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0231] In another embodiment of the present invention, a sugar beet plant or a portion thereof, or pelletized seeds of such a plant, has a genome that enables the development of a beet having 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 through photosynthesis. Suitable varieties for producing sugar beet plants according to this section by introducing resistance according to the present invention are, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0232] Additional embodiments of the present invention relate to sugar beet plants or parts thereof, or pelletized seeds of such plants, in which the genome of the sugar beet plant enables the development of beets having a saccharose concentration of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or even 20%. Suitable varieties for producing sugar beet plants according to this section by introducing resistance according to the present invention are, for example, DAPHNA, KORTESSA KWS, or SABATINA KWS.

[0233] In one embodiment of the present invention, a sugar beet plant or a part thereof, or pelletized seeds of such plant, contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or even at least 30 mutations relative to SEQ ID NOs: 1, 2, or 4.

[0234] A method for producing an organism comprising a mutant of a nucleic acid molecule according to the given embodiment [1] described above, and / or a mutant of a promoter comprising a nucleic acid sequence selected from the group consisting of (a) SEQ ID NO: 7, (b) a nucleotide sequence that hybridizes under stringent conditions with a sequence complementary to the sequence according to (a), and (c) a nucleotide sequence that is at least 70% identical to the sequence according to SEQ ID NO: 7, the method comprising the following steps: (i) A step of preparing an organism or cell containing nucleic acid molecules and / or promoters, (II) A process of increasing the mutation rate of an organism or cell, or of inducing mutagenesis in an organism or cell. (III) A step of performing phenotypic selection of organisms that exhibit a change in resistance or a change in resistance level to Cercospora beticola as a result of mutation, or a step of performing genotypic selection of organisms or cells that include the mutations produced by step (II) in nucleic acid molecules and / or promoters. In addition, optionally, (IV) A process for regenerating an organism from the cells obtained in step (III). Methods that include...

[0235] The organism can be a plant. Preferably, the plant is Beta vulgaris. However, it is also possible to use a single-celled organism as a bacterium. The bacterium may be E. coli. If the organism is a plant, the method can be applied not only in vitro but also in vivo. If the organism is a plant and the method is applied in vitro, a plant cell culture can be established, and an increase in the mutagenesis rate or mutagenesis can be performed in the cell culture. For example, an increase in the mutagenesis rate includes the application of mutagenic agents 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 by precise methods such as gene editing (which will be discussed further below). The regeneration of organisms from cells is described in various standard reference literatures on cell biology. Plant regeneration is described, for example, in the standard reference “Plant biotechnology: comprehensive biotechnology, second supplement” (Michael W. Fowler, Graham Warren, Murray Moo-Young - Pergamon Press - 1992). The regeneration of Beta vulgaris from cell cultures is described by Lindsey & Gallois, “Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens.” Journal of experimental botany 41.5 (1990): 529-536.

[0236] These references also describe methods for establishing plant cell cultures. As further explained above, each promoter of a mutant nucleic acid molecule is preferably characterized by the increased expression rate of the resistance-constituting nucleic acid molecule due to the mutation. Such an effect may also depend on the presence of several mutations. For example, it is possible to introduce two, three, four, five or more mutations into a promoter or nucleic acid molecule.

[0237] Therefore, by introducing mutations, more resistance-constituting proteins can be constructed within the cells, and the proteins have a better effect. This can increase resistance by, for example, at least 1, 2, 3, 4, 5 percent or more compared to a control plant containing the unmodified nucleic acid according to the present invention. The increase can be measured as further described below. Furthermore, resistance due to mutation(s) can increase by at least 1 evaluation score. The determination of the evaluation score is described in other parts of this specification. Furthermore, resistance proteins can confer altered effects as a result of mutations and may in some cases be effective against pathogens adapted to the initial resistance mechanism. In this regard, the present invention also includes such mutant nucleic acid and mutant protein according to the present invention. Preferably, the present invention includes mutants that do not exist in nature and cannot be isolated from nature, ensuring that the pathogen had no opportunity to adapt to such mutants. The above method for producing an organism containing a mutant nucleic acid molecule may further include the step of identifying the organism or respective plant having further increased resistance due to mutation(s). Whether an increase in resistance has occurred can be determined by measuring the evaluation score or resistance level as described herein.

[0238] In addition to the above method of creating an organism containing a mutant nucleic acid molecule or promoter, it is also possible to chemically modify the corresponding nucleic acid in an isolated state to achieve a desired effect (such as those described above). The advantage of this approach is that the compound can be edited more precisely. For this purpose, the following method is provided: A chemically modified nucleic acid molecule according to the above given embodiment [1], and / or (a) SEQ ID NO: 7; (b) A nucleotide sequence that hybridizes with the nucleotide sequence according to (a) under stringent conditions; (c) A nucleotide sequence that is at least 70% identical to the sequence according to SEQ ID NO: 7 A method for creating a chemically modified promoter comprising a nucleotide sequence selected from the group consisting of: the following steps: (I) The step of preparing the above nucleic acid molecule in an isolated form, (II) The following steps: (IIa) Mutagenesis, (IIb) Gene editing, [[ID=2]](IIc) Insertion or deletion of each of restriction and ligation The step of chemically modifying the nucleic acid molecule or promoter by one of comprising the method.

[0239] Furthermore, chemical modifications can be generated by approaches described in other parts of this specification in relation to allele variants. The gene editing shown in step (II) above is equivalent to the term “genome editing.” Optionally, the chemically modified nucleic acid molecule or chemically modified promoter may be subsequently introduced into cells or stably incorporated. Using such cells, the chemically modified nucleic acid molecule and modified promoter can be grown in a cell proliferation state. These can then be isolated in large numbers and expression analysis can be performed. Expression analysis is particularly preferred when the chemical modification relates to a promoter. Cells can be collected and the chemically modified resistance protein can be isolated for chemical analysis. If the cells containing the chemically modified nucleic acid molecule or modified promoter are plant cells, a complete plant can be regenerated from these cells. The approaches described in this paragraph can be carried out following the given methods described above for producing modified nucleic acid molecules and / or modified promoters, and the resulting variants are also part of the present invention. Furthermore, plants containing the chemically modified nucleic acid molecule or modified promoter are also part of the present invention. For this reason, the present invention also relates to plants obtained by this method. Furthermore, the present invention also relates to chemically modified nucleic acid molecules and encoded polypeptides obtained by this method. These compounds may be optimized versions of the original (unmodified) compounds and, as further described above, can increase the resulting resistance level by at least 1, 2, 3, 4, 5 percent or more, or increase the evaluation score by at least 1. In this respect, a method for producing chemically modified nucleic acid molecules is also a method for optimizing nucleic acid molecules. The optimization method may further include the additional step of identifying modified mutants of nucleic acid molecules that result in increased resistance in plants compared to unmodified mutants.

[0240] In further embodiments, the plant of the present invention additionally includes, either transductionally or endogenously, a second nucleic acid molecule encoding a polypeptide capable of conferring resistance to Circospora in the plant expressing the polypeptide. For example, one or more resistance genes or resistance loci described in the prior art can be introduced into the plant by hybridization, transformation, homologous recombination repair, or homologous recombination, provided they are not already present in the initial genotype. For example, these include rhizomania-resistant RZ1 (Lewellen, RT, IO Skoyen, and AW Erichsen, “Breeding sugar beet 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), Feb. 11-12, 1987. IIRB. Secretariat General, 1987) or rhizomania-resistant RZ3 (International Publication No. 2014 / 202044), embodiments relating to RZ3 are described in more detail in item

[94] .

[0241] The present invention further relates to a method for increasing resistance to Circospora in plants of the species Beta vulgaris, wherein the increase in resistance occurs without the presence of the resistance-constituting gene according to the present invention, compared to the same species.

[0242] Increased resistance can be achieved by incorporating the nucleic acid molecule according to the present invention into the genome of at least one cell of a plant of the Beta vulgaris species, and optionally by regenerating the plant from the plant cell. Incorporation can be carried out, for example, by sexual hybridization with one of the Beta vulgaris subsp. maritima described above and subsequent selection, or by homologous recombination repair or homologous recombination. The latter two methods mentioned are preferably supported by site-specific 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, meganucleases, Argonaut nucleases, restriction endonucleases including FokI or its variants, recombinases, or two site-specific nickel endonucleases. Example 1 shows the introduction of a resistance-constituting gene by CRISPR-mediated homologous recombination in Beta vulgaris subsp. vulgaris.

[0243] Furthermore, the present invention relates to a method for agriculturally producing a sugar beet plant of the genus Cercospora that exhibits improved resistance to Cercospora beticola, comprising transferring a chromosomal segment that confers improved resistance to Cercospora beticola into the plant, wherein the chromosomal segment is s4p4293s01 and s4p4295s01 An array represented by a marker selected from a group consisting of, s4p4301s01 and sxh0678s01 The positions are mapped to the positions between the array represented by the marker selected from the group consisting of, A chromosomal segment contains a nucleotide sequence encoding a polypeptide that can confer resistance to Cercospora beticola in plants expressing the polypeptide, wherein the nucleotide sequence is (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 3; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 2; (c) A nucleotide sequence containing a DNA sequence selected from the group consisting of Sequence ID No. 1 or Sequence ID No. 53; (d) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence according to (a), (b), or (c); (e) A nucleotide sequence that encodes a polypeptide 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; (f) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 70% identical to the amino acid sequence of Sequence ID No. 3; (g) A nucleotide sequence that is at least 70% identical to the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 2 This also includes methods characterized by selection from.

[0244] An alternative approach involves increasing the expression of nucleic acid molecules according to the present invention in plants. This can be achieved by modifying the native promoter, which is preferably done using site-specific nucleases, optionally repair models, or gene editing or site-specific mutagenesis. Examples of such nucleases have already been mentioned above. Increased expression of nucleic acid molecules according to the present invention can also be achieved by fusing the nucleic acid molecule with a heterologous promoter that exhibits higher activity compared to the native promoter, particularly after Circospora infection. Fusion can be achieved not only by site-specific nucleases and repair models, but also by direct insertion after double-strand breaks.

[0245] As already mentioned above, methods for increasing circospora resistance can also lead to increased activity and / or stability of polypeptides according to the present invention by modifying the nucleotide sequence of nucleic acid molecules according to the present invention. Such methods for increasing activity by modifying resistance genes are described, for example, in International Publication No. 2006 / 128444 and can be carried out by methods known to those skilled in the art. This approach will be described in further detail below.

[0246] Alternatively, circospora-resistant genotypes can be generated from circospora-sensitive genotypes by random or specific mutagenesis of the nucleic acid sequence of the susceptibility gene, thereby increasing circospora resistance. An example of polymorphisms that distinguish susceptibility alleles from resistance alleles is shown in Figure 1.

[0247] For example, susceptibility alleles can be modified by gene mutations using TALE nucleases (TALENs) or zinc finger nucleases (ZFNs) and the CRISPR / Cas system, as described, for example, in International Publication No. 2014 / 144155 (Manipulation of plant genomes using the CRISPR / Cas system) and Osakabe & Osakabe, Plant Cell Physiol., 56 (2015), 389-400. This can also be achieved using a method called TILLING (Targeted Induced Local Lesions in Genomes), for example, in German Patent Application Publication No. 102013101617, a method for inducing point mutations in susceptibility genes and subsequently selecting plants exhibiting desirable, i.e., resistance-constituting mutations, such as barley resistant to stripe dwarf virus. See paragraphs

[0014] ,

[0026] , and

[0038] on pages 4, 8, and 12 of German Patent Application Publication No. 102013101617. The TILLING method is also described in detail in the publication by Henikoff et al. (Henikoff et al., Plant Physiol. 135, 2004, 630-636).

[0248] These methods preferably result in an improvement in resistance of at least one evaluation score, and more preferably, an improvement in resistance of at least two, three evaluation scores or more. Plants exhibiting one or more mutations in endogenous nucleic acid molecules, as shown in Figure 1, can be identified after mutagenesis of plant cells and subsequent regeneration of plants from the mutageneised plant cells, or after mutagenesis of plants. In this regard, the plants according to the present invention described above may be characterized by an increase in resistance of at least one evaluation score, preferably at least two evaluation scores or more. Alternatively, the resistance of plants according to the present invention can be increased by, for example, at least 1, 2, 3, 4, 5 percent or more compared to control plants without the nucleic acid according to the present invention. The increase can be measured by inoculating one healthy leaf each with an isolate of the pathogen and determining the affected surface after 15 days. A 5% reduction in the affected surface corresponds to a 5% increase in resistance. Further parameters for performing the measurement can be derived from the “resistance test” of the given embodiment below.

[0249] An additional embodiment of the present invention is a method for producing circospora-resistant plants, which can be done by transforming plant cells with nucleic acid molecules, recombinant DNA molecules, or vectors or expression cassettes according to the present invention and regenerating transgenic plants from the transformed plant cells (see Example 2), as well as by generating circospora-resistant genotypes by random or targeted mutagenesis of the nucleic acid sequence of susceptibility genes, as described above, or by hybridization and selection using, for example, one of the Beta vulgaris subsp. maritima described above. The vector or expression cassette and the method for transforming plants have already been described above.

[0250] Alternatively, a method for producing circospora-resistant plants involves introducing a site-specific nuclease and a repair matrix into cells of the Beta vulgaris species plant, as described above, wherein the site-specific nuclease can generate at least one double-strand break in the DNA in the cell's genome, preferably upstream and / or downstream of the target region, and the repair matrix comprises nucleic acid molecules according to the present invention. The method further comprises culturing these cells under conditions that allow homologous recombination repair or homologous recombination, so that the nucleic acid molecules are incorporated from the repair matrix into the plant's genome. Furthermore, it encompasses regenerating plants from the modified plant cells (see Example 1).

[0251] In a preferred embodiment, the target region is an allele variant of the nucleic acid molecule according to the present invention, the allele variant encoding a polypeptide that does not confer resistance to circospora. In a further preferred embodiment, the allele variant comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6 and / or a coding DNA sequence of SEQ ID NO: 5 or a genomic DNA sequence of SEQ ID NO: 4.

[0252] As described in relation to the nucleic acid molecule according to the present invention, substitutions, deletions, insertions, additions, and / or any other changes may be introduced, either alone or in combination, that actually alter the nucleotide sequence but perform the same function as the original sequence, in this case the nucleotide sequence of the allele variant of the nucleic acid molecule according to the present invention. Accordingly, in further embodiments, the present invention includes a nucleotide sequence that is a derivative of a polypeptide encoded by the allele variant of the nucleic acid molecule according to the present invention, or a polypeptide comprising the amino acid sequence of the allele variant of the nucleic acid molecule according to the present invention. An induced amino acid sequence having at least one substitution, deletion, insertion, or addition of one or more amino acids, while retaining the functionality of the encoded polypeptide / protein, is a polypeptide derivative. Thus, the nucleotide sequence can be introduced, either alone or in combination with a gene, by conventional methods known in the prior art, such as site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, genome editing, etc., which actually alter the nucleotide sequence but perform the same function as the original sequence.

[0253] With respect to the amino acid sequence, it has a common structural domain and / or common functional activity after modification by the method described above. A nucleotide sequence or amino acid sequence that is at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least approximately 100% identical to the nucleotide sequence or amino acid sequence of the allele variant of the nucleic acid molecule according to the present invention as described herein is defined herein as sufficiently similar. Accordingly, the present invention includes a nucleotide sequence that can hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the nucleotide sequence or corresponding amino acid sequence of the allele variant of the nucleic acid molecule according to the present invention.

[0254] In a further preferred embodiment, the method according to the present invention is characterized in that a double-strand break occurs in the allele variant of the nucleic acid molecule according to Embodiment [1], or at least one double-strand break occurs at a position at least 10,000 base pairs upstream or downstream of the allele variant encoding a polypeptide that does not confer resistance to circospora.

[0255] Because numerous different susceptibility sequences may arise from the nucleic acid molecule according to the present invention but do not confer resistance to circospora, the sequences listed above (SEQ ID NOs: 4, 5, and 6) should be considered merely examples of sequences, and it will be obvious to those skilled in the art that the present invention is not limited to allele variants of the nucleic acid molecule according to the present invention as described above. Such allele variants are, (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 6; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 5; (c) Nucleotide sequence containing DNA sequence according to Sequence ID No. 4; (d) A nucleotide sequence that hybridizes under stringent conditions with the complementary sequence according to (a), (b), or (c); (e) A nucleotide sequence that encodes a polypeptide 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; (f) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6; (g) A nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the DNA sequence by SEQ ID NO: 4 or SEQ ID NO: 5. It may include a nucleotide sequence selected from the following.

[0256] As described above, quantitative inheritance of QTLs often introduces not only desired resistance to plants, but also undesirable traits, such as reduced yield, through the inheritance of additional genes unrelated to the positive trait of resistance. This is increasingly common when resistance is inherited in existing cultivars by many resistance genes with only slight effects, as in the case of Circospora resistance. Therefore, in preferred embodiments, the introduction of nucleic acid molecules, vectors, or expression cassettes according to the present invention, which already exhibit a dominant resistance effect, is not associated with the introduction of undesirable traits, and yield is preferably not adversely affected. Furthermore, plants obtained by such methods are included in the present invention.

[0257] While conventional QTL analysis techniques have been able to detect actual QTLs, the underlying genomic regions that exhibit QTL effects also mediate the aforementioned disadvantages, leading to the discussion of "linkage drugs." Simultaneously, QTLs and their associated effects have not been uniformly described in each conventional technique, and only weak effects have been mediated. Therefore, the application of these results to the breeding of circospora-resistant plants has been limited and largely uncertain. The identification of resistance genes described herein now enables targeted breeding of sugar beets and the controlled incorporation of resistance genes into their gene pool. This ensures the breeding and production of entirely new circospora-resistant cultivars that exhibit high resistance to the pathogen without negatively impacting sugar yield.

[0258] The present invention also relates to a method / process for identifying, detecting, and optionally selecting and / or providing plant species of Beta vulgaris resistant to the pathogen Circospora, wherein the method comprises a step of detecting the presence and / or expression of nucleic acid molecules or polypeptides according to the present invention in a plant or a sample / part thereof. The presence and / or expression of nucleic acid molecules or polypeptides according to the present invention can be tested by standard methods known to those skilled in the art, e.g., PCR, RT-PCR or Western blotting. Preferably, during PCR, the oligonucleotide or primer hybridizes with a genomic template containing a nucleotide sequence defined in step (i) of

[0104] at a method / distance such that the resulting amplified product consists of 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. The identified / detected and optionally selected and / or provided plants may be plants containing the resistance gene according to the present invention, and further containing the nucleotide sequence by SEQ ID NO: 182 and / or the resistance allele by the marker s4p8772s01, or plants that can be obtained from seeds deposited at the NCIMB in Aberdeen, UK, under access number NCIMB 43646. The methods and processes described in this section may also be used to exclude or ignore plants that do not possess the resistance gene according to the present invention. This is particularly useful, for example, in breeding programs where some but not all of the offspring plants are resistant.

[0259] Furthermore, the identification method according to the present invention also includes detecting the nucleic acid molecule according to the present invention by detecting at least one polymorphism between a resistant sequence and a susceptible sequence, i.e., between the sequence of the nucleic acid molecule according to the present invention and the sequence of an allele variant of the nucleic acid molecule according to the present invention, using one or more molecular markers that detect polymorphisms. As already mentioned above, it is obvious to those skilled in the art that there are many susceptible sequences, i.e., many sequences that encode allele variants of the nucleic acid molecule according to the present invention. As an example of sequence comparison with the nucleotide sequence of the nucleic acid molecule according to the present invention, one of these is shown in Figure 1. A preferred embodiment of the method according to the present invention includes detecting at least one polymorphism shown in Figure 1 using a molecular marker that detects polymorphisms, in particular diagnostic polymorphisms. This detection is preferably performed using at least one molecular marker for one polymorphism, in particular one diagnostic polymorphism. It is known to those skilled in the art which marker techniques should be applied to detect the corresponding polymorphisms and how molecular markers for this purpose should be constructed (see Advances in Seed Science and Technology Vol. I, Vanangamudi et al., 2008). Furthermore, the present invention encompasses the use of molecular markers that describe or detect polymorphisms as shown in Figure 1, for example, molecular markers for detecting polymorphisms as shown in Figure 1. This also makes it possible to use markers that do not distinguish between various polymorphisms, insofar as the markers can detect polymorphisms that occur in nucleic acid molecules according to the present invention but are not present in susceptible allele variants.

[0260] Alternatively or additionally, an identification method according to the present invention includes the step of detecting at least one marker locus in the nucleotide sequence of a nucleic acid molecule according to the present invention or in its co-separated region. Preferably, the co-separated region is a circospora resistance conferred by the polypeptide according to the present invention, or a genomic region in Beta vulgaris that co-separates with the nucleic acid molecule according to the present invention, and more preferably, the co-separated region includes and adjacent to the markers sxh0678s01 and s4p0264s01, markers s4p4301s01 and s4p2271s01, markers s4p4301s01 and s4p4293s01, or markers s4p4301s01 and s4p4295s01. Thus, detection can be carried out by a method step in which at least one marker or at least one primer pair is bound at the locus according to SEQ ID NO: 74 or 75, preferably the locus according to SEQ ID NO: 76 or 77, and optionally a signal, such as a fluorescent signal or a sequence amplification, is generated as a result. Therefore, alternatively or additionally, the co-separated region may include the sequence of sequence numbers 74 and / or 75, or sequence numbers 76 and / or 77. Furthermore, the aforementioned identification method is also a method for selecting plants that exhibit resistance to Circospora according to the present invention. The selection method includes a final step of selecting resistant plants.

[0261] In connection therewith, the present invention also includes the development or production of molecular markers suitable for detecting the above-mentioned polymorphism between the nucleic acid molecule (resistance allele) and the susceptibility allele mutant according to the present invention, the markers being suitable for detecting the polymorphism shown in Figure 1, constructing hybridization probes that specifically bind to the nucleotide sequence of the nucleic acid molecule according to the present invention, or amplifying regions specific to the nucleic acid molecule according to the present invention in PCR, and thus for producing nucleic acid molecule pairs suitable for detecting them in plants or plant cells.

[0262] The present invention includes a method for producing nucleic acid molecule pairs, preferably in the form of oligonucleotides, which are suitable for attaching to a region specific to the nucleic acid molecule according to the present invention as forward and reverse primers and amplifying them in polymerase chain reaction (PCR), or suitable for hybridizing to a region in the Beta vulgaris genome that has circospora resistance conferred by the polypeptide according to the present invention in Beta vulgaris or co-separation with the nucleic acid molecule according to the present invention, preferably as forward and reverse primers, preferably as oligonucleotides. Examples of primers suitable for detecting resistance-mediated nucleotide sequences according to the present invention are shown in SEQ ID NOs: 98 and 99. These two sequences construct a primer pair that can be used for PCR. The present invention also includes a kit containing an oligonucleotide or molecular marker according to the present invention.

[0263] A method for producing oligonucleotides includes, first, comparing the nucleotide sequence of a nucleic acid molecule according to the present invention with the nucleotide sequence of a corresponding nucleic acid molecule that does not confer resistance, or a susceptible allele mutant having a nucleotide sequence according to Sequence ID No. 4 or 5; identifying the sequence difference between the two nucleotide sequences; and generating a nucleic acid molecule that specifically binds to the nucleic acid molecule according to the present invention but does not bind to the nucleic acid molecule that does not mediate resistance, i.e., an oligonucleotide in this case.

[0264] Furthermore, the oligonucleotides according to the present invention can be conjugated to a fluorescent dye to generate a fluorescent signal, for example, under excitation with light of a corresponding wavelength. The fluorescent dye may be fluorochrome. The oligonucleotides according to the present invention may be coupled with other compounds suitable for signal generation. Such oligonucleotides do not exist in nature and cannot be isolated from nature. To prepare such labeled oligonucleotides, the following can be done: DNA can be bioorthogonally labeled. For this purpose, DNA can be labeled in vivo or in vitro with a nucleoside analog, which can then be coupled, for example, to a fluorophore by a Staudinger reaction. In addition, fluorophores may be chemically added to DNA. Oligonucleotides can be labeled by phosphoramidite synthesis with fluorophores used, for example, in QPCR, DNA sequencing, and in situ hybridization. Furthermore, DNA may be enzymatically generated using fluorescent nucleotides in the course of a polymerase chain reaction, or labeled by ligase or terminal deoxynucleotidyltransferase. DNA can also be detected indirectly by biotinylation and fluorescent avidin. For coupling, fluorescein, 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, oligonucleotides with FAM labeling, especially primers, are included in the present invention. FAM is preferably present as 6-FAM, but other FAM variants, such as 5-FAM, may also be used depending on the desired wavelengths of emission and excitation. Examples of additional fluorescent markers are AlexaFluor, ATTO, Dabcyl, HEX, Rox, TET, Texas Red, and Yakima Yellow. Depending on the application, oligonucleotides may have base or sugar phosphate spine modifications.In particular, amino-dT, azide-dT, 2-aminopurine, 5-Br-dC, 2'-deoxyinosine (INO), 3'-deoxy-A, C, G, 5-Met-dC, 5-OH-Met-dCN, and 6-Met-dA are included in these categories.

[0265] Furthermore, the present invention also relates to a marker chip ("DNA chip", "assay", or microarray) comprising at least one oligonucleotide according to the present invention that is suitable for detection. The marker chip is suitable for application to one or more detection methods according to the present invention.

[0266] The present invention also includes a method for producing a protein according to the present invention. This method comprises preparing or culturing a cell culture containing SEQ ID NO: 2, and subsequently expressing the protein encoded by SEQ ID NO: 2.

[0267] Furthermore, the present invention also relates to circospora-resistant plants or a portion thereof identified and, where applicable, selected by the methods described above. In particular, the present invention relates to a population of plants comprising plants obtained by one of the methods according to the present invention described above, preferably resistant to circospora leaf spot disease or circospora invasion, and characterized by the presence of nucleic acid molecules according to the present invention. The population preferably has at least 10, preferably at least 50, more preferably at least 100, particularly preferably at least 500, and especially in agricultural cultivation, preferably at least 1000 plants. The proportion of plants in the population that do not possess the nucleic acid molecules according to the present invention and / or are susceptible to circospora leaf spot disease 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.

[0268] The detailed mapping described above allowed us to determine the location of the circospora resistance-conferring gene in the genome of Beta vulgaris subsp. maritima, and to identify the gene itself and the surrounding sequence region. This forms the basis for the development of DNA hybridization probes or genetic markers in the target region, which can be used to detect the circospora resistance-mediated gene or to distinguish it from genes that do not confer resistance.

[0269] DNA hybridization probes are derived from the sequences of circospora resistance-constituting genes and can be used to screen genome banks and / or cDNA banks of desired organisms. The probes can be used to amplify the identified homologous genes by a known polymerase chain reaction (PCR) process to determine whether the circospora resistance-constituting gene is endogenously present in the organism or successfully introduced as a heterologous element.

[0270] Here, conventional hybridization, cloning, and sequencing methods can be used by those skilled in the art, as cited, for example, in 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 circospora resistance-constituting gene. To achieve specific hybridization, it is desirable that such probes be specific and have a length of at least 15 nucleotides, preferably at least 20 nucleotides. Detailed guides to 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, Ausubel et al., eds., Greene Publishing and Wiley Interscience, New York (1995).

[0271] Therefore, nucleic acid molecules having a length of at least 15, 16, 17, 18, 19, or 20 nucleotides, preferably at least 21, 22, 23, 24, or 25 nucleotides, particularly preferably at least 30, 35, 40, 45, or 50 nucleotides, and particularly preferably at least 100, 200, 300, 500, or 1000 nucleotides, are the subject of the present invention, and these nucleic acid molecules specifically hybridize with the aforementioned nucleotide sequences according to the present invention, including circospora resistance-constituting genes. This explicitly includes the range of 15 to 35 nucleotides.

[0272] Therefore, the present invention also relates to oligonucleotides, particularly markers as primer oligonucleotides. These include nucleic acid molecules of at least 15 nucleotides in length that specifically hybridize with the nucleotide sequence defined above.

[0273] In particular, the present invention comprises a kit containing a pair of nucleic acid molecules, preferably in the form of an oligonucleotide, or a pair of such oligonucleotides, which are suitable as forward and reverse primers for hybridization with regions specific to the nucleic acid molecules according to the present invention and for amplification thereof in polymerase chain reaction (PCR), or for hybridization with regions in the Beta vulgaris genome that exhibit circospora resistance or co-separation with the nucleic acid molecules according to the present invention, as conferred by the polypeptide according to the present invention in Beta vulgaris.

[0274] The following advantages of breeding and developing new resistant plant lines of the genus Bhutia can also be achieved by the present invention. Sequence information and the identification of polymorphisms that enable the distinction between resistance alleles and susceptibility alleles of the disclosed gene, i.e., between alleles that confer circospora resistance and alleles that do not confer this resistance, enable the development of markers directly within the gene, as well as in upstream and downstream regions, as described above. This is an important convenience for plant breeders, particularly with regard to the development of optimized superior lines without "linkage drugs." Furthermore, knowledge of sequence structure can be used to identify additional resistance genes, particularly against circospora, that are homologous or orthologous, for example.

[0275] Accordingly, the present invention also encompasses methods for identifying additional nucleic acid molecules encoding polypeptides or additional proteins that can confer resistance to circospora in plants expressing the polypeptide. This allows those skilled in the art to use databases with suitable search profiles and computer programs for homologous sequence screening or sequence comparison. Furthermore, those skilled in the art can induce additional DNA sequences encoding circospora-resistant proteins using conventional molecular biological techniques and use these 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 used for screening genome banks and / or cDNA banks of desired organisms. Here, those skilled in the art can use conventional hybridization, cloning, and sequencing methods, which 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. Those skilled in the art can also synthesize and use oligonucleotide primers to amplify the sequences of circospora-resistant nucleic acid molecules using known sequences.

[0276] Accordingly, in one embodiment, the present invention comprises a method for identifying a nucleic acid molecule encoding a polypeptide that can confer resistance to circospora in a plant of the Beta vulgaris species in which the polypeptide is expressed. Thus, the method comprises comparing the amino acid sequence of the polypeptide according to the present invention that confers circospora resistance in Beta vulgaris subsp. vulgaris with an amino acid sequence from a sequence database or the sequence of an allele variant of the polypeptide according to the present invention in a genotype of the Beta vulgaris species. Furthermore, the method according to the present invention comprises identifying an amino acid sequence or allele variant that is at least 80% identical to the amino acid sequence of the polypeptide according to the present invention, introducing the nucleic acid molecule encoding the identified amino acid sequence or allele variant into a plant of the Beta vulgaris species; expressing the nucleic acid molecule in the plant; and optionally subsequently verifying resistance to circospora.

[0277] As described above, additional circospora resistance-constituting proteins or their coding genes, i.e., homologs, analogs, and orthologues, that are at least 70%, preferably at least 80%, particularly preferably at least 90%, particularly 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 present invention, can be identified by classical bioinformatics approaches (database searches and computer programs for screening homologous sequences).

[0278] Therefore, the term homolog typically means that the genes in question (derived from two different plant species) have essentially the same function and a common ancestor, and therefore exhibit significant identity in their nucleic acid sequences or encoded amino acid sequences. However, many genes are homologous to each other even if their protein sequences do not produce a significant paired alignment. In contrast, the term analog describes genes or proteins that have the same or similar function but are not made from the same structure, i.e., do not share a common ancestor. In this case, significant identity cannot often be established in their nucleic acid sequences or encoded amino acid sequences, or, in the best case, in specific functional domains.

[0279] In relation to genome sequencing, homologs are further classified for annotation purposes. The terms orthology and paralogy are introduced for this purpose. Orthologs are genes associated by speciation events. Paralogs are genes derived from duplication events.

[0280] Therefore, a gene is essentially a homolog, analog, or ortholog in the sense of the present invention if it can confer circospora resistance in plants. For confirmation, methods already known to those skilled in the art, such as PCR amplification of the identified homolog, analog, or ortholog, cloning into an expression vector, introduction into a target plant or plant cell, and confirmation of resistance are used.

[0281] As described above, the use of alleles of resistance genes in cisgenic or transgenic approaches as disclosed herein opens up the possibility of new resistant species of the genus Chamaecyparis, which can exhibit higher resistance using dose-effects or optimize resistance expression by avoiding resistance disruption through stacking of the disclosed gene with other resistance genes. Gene modification by tilling or targeted operations is also possible to optimize codon selection and increase or develop the expression of novel or modified resistance alleles. According to preferred embodiments, codon-optimized sequences or modified resistance alleles are not naturally occurring and are artificial. An example of a modified genome sequence is shown in SEQ ID NO: 94, where codons at positions 16-18 are modified, but the encoded amino acid sequence remains unchanged, corresponding to SEQ ID NO: 3. An example of a modified cDNA sequence is shown in SEQ ID NO: 95, where codons at positions 55-57 are modified, but the encoded amino acid sequence remains unchanged, corresponding to SEQ ID NO: 3. SEQ ID NOs. 94 and 95 are also examples of hybridization sequences. An example of a modified resistance-constituting allele is shown in the amino acid sequence of SEQ ID NO: 96, where the amino acid valine at position 209 is replaced with the amino acid leucine. The amino acid sequence of SEQ ID NO: 96 is encoded by the modified cDNA of SEQ ID NO: 97. These sequences do not exist in nature and are artificial. For example, when replacing amino acids in the resistance-mediated sequence of SEQ ID NO: 3, the following amino acid substitutions are recommended: a) Glycine, alanine, valine, leucine, isoleucine b) Serine, cysteine, selenocysteine, threonine, methionine c) Phenylalanine, tyrosine, tryptophan d) Histidine, lysine, arginine e) Aspartic acid, glutamic acid, asparagine, glutamine.

[0282] The present invention also relates to the use of alleles of identified circospora resistance-constituting genes in plants in gene stacks or molecular stacks with other genetic elements that can confer agronomically advantageous characteristics. This can significantly increase the economic value of cultivated plants, for example, by increasing yield compared to plants with the same genetic characteristics but without the nucleic acid constituting according to the present invention. Furthermore, it may open up new cultivation areas for these plants that were previously unsuitable for cultivation due to biological factors such as high pathogen pressure. In particular, the present invention relates to the use of alleles of identified circospora resistance-constituting genes in methods for controlling the invasion of the pathogen circospora beticola in agricultural or horticultural cultivation of plants of the genus Bhut, including, for example, identifying and selecting plants of the genus Bhut using one of the methods described above, and / or cultivating such selected plants or their offspring. Accordingly, the present invention includes a method for cultivating Beta vulgaris plants, comprising: a first step of preparing a circospora-resistant plant of the Beta vulgaris species according to the present invention, or producing a plant of the Beta vulgaris species using the production method according to the present invention, or identifying and selecting a plant of the Beta vulgaris species using the identification method according to the present invention described above; and a second step of cultivating the plant from the first step, or sowing a seed stock of the plant from the first step, or growing the plant from the first step. The cultivation method thereby prevents circospora invasion of the cultivated plant. The cultivation method may be part of a sugar production method. The sugar production method includes the steps of the cultivation method and further includes harvesting the cultivated plant as the second to last step and extracting sugar from the aforementioned plant as the final step. The identification or detection methods, markers, and oligonucleotides described herein can be used, for example, for seeds deposited at the NCIMB in Aberdeen, UK, under access number NCIMB 43646, or for plants obtained from deposited seeds.Furthermore, these can be used to distinguish between a) plants that are descendants of deposited seeds, b) plants into which the resistance gene according to the present invention has been introduced by one of the methods described herein (e.g., transformation), or c) plants containing the resistance gene according to the present invention and plants lacking the resistance gene according to the present invention.

[0283] The cultivation method may be part of the method for producing a seed stock. The method for producing a seed stock includes the steps of the cultivation method, and further includes vernalizing the cultivated plants as the second to last step, and extracting seeds from the aforementioned plants as the final step.

[0284] The extracted seeds can be optionally pelletized to obtain a pelletized seed stock of the Beta vulgaris species. In this example, this is a method for preparing a pelletized seed stock.

[0285] Furthermore, the method for producing seed stocks can be designed as a method for producing circospora-resistant seed stocks. The method for producing circospora-resistant seed stocks includes the steps of the above method for producing seed stocks, and as a final step, verifying nucleic acids according to the method herein in at least one of the extracted seeds, preferably at least 0.1% or at least 1% of the extracted seeds. The verification is performed so that the seeds remain viable, particularly preferably without rendering them viable. That is, the extraction of DNA required for verification from the seeds does not render them viable. In such an example, the verification of nucleic acids according to the method may be performed on a particularly large proportion of all extracted seeds. For example, verification may be performed on at least 2%, preferably at least 3%, and particularly preferably at least 4% of all extracted seeds.

[0286] The plants, their cells, or seeds or seed stocks according to the present invention possess or can be conferred additional agronomically advantageous properties. One example is tolerance or resistance to herbicides such as glyphosate, glufosinate, or ALS inhibitors. Tolerance to glyphosate or ALS inhibitor herbicides is preferred. A specific embodiment of glyphosate resistance is disclosed in U.S. Patent No. 7,335,816. Such glyphosate resistance can be obtained, for example, from seed stocks stored at NCIMB in Aberdeen, Scotland, with access numbers NCIMB 41158 or NCIMB 41159. Glyphosate-resistant sugar beet plants can be obtained using such seeds. Glyphosate resistance can also be introduced into other species of the genus Bhutella by hybridization.

[0287] Therefore, in relation to glyphosate resistance, the present invention contains nucleic acids according to the present invention, and further, a) A DNA fragment of the genomic DNA of a plant, a part thereof, or a seed can be amplified by a polymerase chain reaction using 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 b) A DNA fragment of the genomic DNA of a plant, a part thereof, or a seed can be amplified by a polymerase chain reaction using 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%, to the nucleotide sequence of SEQ ID NO: 86, and / or c) A DNA fragment of the genomic DNA of a plant, a part thereof, or a seed can be amplified by a polymerase chain reaction using 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%, to the nucleotide sequence of SEQ ID NO: 89. This also includes plants, their cells, or seeds or seed stocks, characterized by the above.

[0288] In certain embodiments, the wild-type Beta vulgaris epsp synthase has an amino acid sequence provided in the NCBI reference sequence XP_010692222.1. This wild-type epsp synthase may be mutated or modified (e.g., by one of the mutation or modification methods disclosed herein) to confer resistance to glyphosate. Such a mutation may result in 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. Plants comprising the mutated epsp synthase and the resistance gene according to the present invention are included herein. This includes the resistance gene of the present invention and further having an amino acid different from proline at position 179, i) Array of sequence number 223, ii) Sequences having an amino acid different from serine and proline at position 179 i), or iii) A sequence having at least 90%, more preferably 95%, and most preferably 99% identity with sequence i) or ii) throughout its entire length. The plant includes a nucleic acid molecule encoding epsp synthase, which contains an amino acid sequence selected from the above.

[0289] In this regard, mutant EPSP synthase may be encoded by a nucleic acid sequence, for example, SEQ ID NO: 224. Further technical details regarding the use of mutant EPSP synthase can be obtained from International Publication No. 2020064687. Plants containing the resistance gene according to the present invention and expressing mutant EPSP synthase may further possess resistance to one or more ALS inhibitor herbicides described herein. Plants containing the resistance gene according to the present invention and expressing mutant EPSP synthase may be non-transgenic plants.

[0290] In relation to the present invention, mutant EPSP synthase is an artificial compound that does not exist in nature. According to certain embodiments, a plant containing mutant EPSP synthase and the resistance gene according to the present invention is not essentially a product of a biological process.

[0291] Specific embodiments of ALS inhibitor herbicide resistance are disclosed in International Publication No. 2012 / 049268. For example, such ALS inhibitor herbicide resistance can be obtained from a deposit at NCIMB No. 41705 in Aberdeen, UK. Furthermore, such ALS inhibitor resistance can be induced by tilling or site-directed mutagenesis, such as gene editing using CRISPR / Cas, CRISPR / Cpf1, TALEN, or zinc finger nucleases. Accordingly, the present invention also includes plants, their cells, or seeds or seed stocks containing nucleic acids according to the present invention and further characterized by exhibiting a mutation in the endogenous acetolactate synthase gene, wherein the acetolactate synthase gene encodes an acetolactate synthase protein having an amino acid different from tryptophan as a result of the 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 by position 569 of Sequence ID No. 90. Furthermore, the specific sequence of the mutant acetolactate synthase gene of Sequence ID No. 91 is preferred. The mutant sequence of the acetolactate synthase gene, i.e., the sequence of Sequence ID No. 91, does not exist in nature and cannot be isolated from nature. Furthermore, the mutation may exist both heterozygously and homozygously in plants, their cells, or seeds or seed stocks. The presence of a homozygous mutation is preferred because it promotes the development of a more stable or more concentrated resistant phenotype.

[0292] Numerous additional herbicides and their applicability are known to those skilled in the art from the prior art. Those skilled in the art can use the prior art to gain knowledge of which genetic elements to use and in what manner to achieve the corresponding resistance in plants.

[0293] Furthermore, herbicide resistance has a synergistic effect, as the use of herbicides reduces weed growth. This is advantageous in combating Circospora because the conidia (asexual spores) or pseudospores (mycelia) of Circospora beticola are known to survive on plant material for up to two years.

[0294] A further example of an agronomically advantageous trait is additional pathogen resistance, where the pathogen may be, for example, an insect, virus, nematode, bacterium, or fungus. For example, broad pathogen defense in a plant can be achieved by different combinations of pathogen resistance / tolerance, as the genetic elements may exhibit additive effects on each other. For example, numerous resistance genes for this are known to those skilled in the art as genetic elements. For example, U.S. Patent Application Publication No. 20160152999 discloses a RZ resistance gene for root rot, a disease caused by the causative agent "beet necrotic yellowing of the leaf vein virus." Several disease resistances contained in a single plant can have synergistic effects on each other. When a plant is first invaded by a pathogen, its immune system is usually weakened, and the epidermis, which acts as an outer barrier, is often damaged, increasing the likelihood of further infection. An additional example of an agronomically advantageous trait is cold tolerance or frost tolerance. Plants exhibiting this characteristic can be sown earlier in the year or left in the field longer, which may result in increased yields, for example. Those skilled in the art may also use prior art to find suitable genetic elements. Additional examples of agronomically advantageous characteristics include water use efficiency, nitrogen use efficiency, and yield. Genetic elements that can be used to confer such characteristics can be found in the prior art.

[0295] Furthermore, numerous modifications for pathogen defense are known to those skilled in the art. In addition to the R gene family, which is often described, the Avr / R approach, Avr gene complementation (International Publication No. 2013 / 127379), R gene self-activation (International Publication No. 2006 / 128444), or the HIGS (host-induced gene silencing) approach (e.g., International Publication No. 2013 / 050024) can be advantageously used. In particular, R gene self-activation may be important to the present invention. For this purpose, nucleic acids encoding self-activating resistance proteins are constructed to produce resistance to pathogens in plants. In this case, the nucleic acid has only a limited portion of an NBS-LRR resistance gene, such as the wb-R gene, extending from the 5' end of the coding region of the NBS-LRR resistance gene to the beginning of coding in the NBS domain of the downstream NBS-LRR resistance gene.

[0296] In this regard, the method also includes a step of removing a region of nucleic acid according to the present invention that encodes the N-terminal region, starting at the p-loop of the NBS domain and extending to the end of the N-terminal region.

[0297] Resistance proteins encoded by such truncated nucleic acids are generally self-activating in that they induce an immune response in plants even in the absence of the relevant pathogen, thereby enhancing the plant's basal immunity. Furthermore, the present invention encompasses such truncated nucleic acids and polypeptides encoded thereby.

[0298] Furthermore, the present invention also includes the use of alleles of the circospora resistance-constituting gene identified in the above-described manner for combination with one of the aforementioned modifications or the aforementioned genetic elements that can lead to one or more agronomically advantageous traits in a plant.

[0299] In addition to relating to plants according to the present invention, the present invention also relates to materials or substances for the chemical industry, such as seeds or offspring, or their organs, plant parts, tissues or cells, in the production of products, such as food and animal feed, preferably sugar or syrup (molasses) (wherein molasses is also used for industrial purposes), in the production of alcohol, for example, or as growing media for the production of biotechnology products, such as refined chemicals, pharmaceuticals or their precursors, diagnostic agents, cosmetics, bioethanol or biogas. An example of the use of sugar beets as a bio-based raw material in a biogas plant is described in German Patent Application Publication No. 102012022178. See, for example, paragraph 10.

[0300] The following examples illustrate the present invention but do not limit its subject matter. Unless otherwise indicated, standard molecular biological methods were used. 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, eds., Academic Press, London, 1987; and Weir et al., Handbook of Experimental Immunology, Volumes I-IV, Blackwell, eds., 1986.

[0301] Some of the most important sequences according to the present invention are described in detail below: -Sequence ID 1: Genomic DNA sequence of the circospora resistance-conferring gene derived from Beta vulgaris subsp. maritima -Sequence ID 2: cDNA sequence of the circospora resistance-conferring gene, which does not exist in nature. -SEQ ID NO: 3: Amino acid sequence of the circospora resistance-conferring protein encoded by SEQ ID NO: 1 or SEQ ID NO: 2 -SEQ ID NO: 4: Genomic DNA sequence of a susceptible mutant of the circospora resistance-conferring gene -SEQ ID NO: 5: cDNA of a susceptible mutant of the circospora resistance-conferring gene -SEQ ID NO: 6: Amino acid sequence of a susceptible mutant of the circospora resistance-conferring gene -Sequence ID 7: Native promoter of the circospora resistance-conferring gene derived from Beta vulgaris subsp. maritima - Sequence ID 8: Native terminator of the circospora resistance-conferring gene derived from Beta vulgaris subsp. maritima - Sequence ID 53: Sequence of the gene locus derived from Beta vulgaris subsp. maritima, which contains the gene that confers circospora resistance according to Sequence ID 1.

[0302] Examples Example 1: Introduction of resistance-constituting genes using CRISPR-mediated homologous recombination in Beta vulgaris subsp. vulgaris crRNA design and selection: We designed crRNAs suitable for Cpf1-mediated induction of double-strand breaks using CRISPR RGEN Tools (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)). For this purpose, we searched for suitable protospacers within genomic DNA sequences of 500-1300 bp in length adjacent to the 5' and 3' ends of the circospora resistance gene derived from Beta vulgaris subsp. maritima. To ensure the functionality of the endonuclease Cpf1 derived from the Lacnospiraceae bacterium ND2006(Lb), a 24nt protospacer was selected, with a 5'-terminal genome-binding sequence flanked by an essential protospacer-adjacent motif (PAM) having the sequence 5'-TTTV-3' (V=G, C, or A). Suitable protospacers were selected according to the tool's specified quality criteria and adjusted for potential off-targets using the reference genome of B. vulgaris subsp. vulgaris. For serial testing, only crRNAs containing up to 15 identical bases, including the functional PAM, were selected in addition to the actual target sequence.The first 18nt of the protospacer is essential for the detection and cleavage of the target sequence, thus preventing undesirable cleavage within other genomic sequences (Tang, X., LG Lowder, T. Zhang, AA Malzahn, X. Zheng, DF Voytas, Z. Zhong, Y. Chen, Q. Ren, Q. Li, ER Kirkland, Y. Zhang, and Y. Qi (2017), “A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants.” Nat Plants 3: 17018). In this way, we were able to identify four potential crRNAs (5'crRNA#1~4) in the 5'-flanking region and three crRNAs (3'crRNA#1~3) in the 3'-flanking region of the resistance gene (see Table A).

[0303] [Table 3]

[0304] Cloning of genetic elements: For the cloning of the cpf1 expression cassette and crRNA expression cassette, the detection sequence of the restriction enzyme BbsI, which hinders cloning, was first removed from the target vector pZFNnptII by introducing a point mutation (T→G). Mutagenesis was performed using two mutagenesis primers (see Table B) and a mutagenesis kit according to the manufacturer's specifications.

[0305] [Table 4]

[0306] To express the Lbcpf1 gene in B. vulgaris, a codon-optimized DNA sequence from A. thaliana, containing a 5'-flanking PcUbi promoter sequence (SEQ ID NO: 79) derived from Petroselinum crispum and a 3'-flanking 3A terminator sequence derived from pea, was synthetically constructed as a DNA fragment. To avoid unintended cleavage within the coding region, the cloning-related restriction interface (HindIII) in the Lbcpf1 coding sequence (CDS) [SEQ ID NO: 78] was removed by introducing silent mutations (base exchanges that do not alter the amino acid sequence). Codon optimization was performed using the GeneArt algorithm of Invitrogene / ThermoScientific. To enable Cpf1 transport within the cell nucleus, the coding sequence of the nuclear position signaling (NLS) of SV40 was incorporated into the CDS of cpf1 at its 5' end, and the NLS of nucleoplasmin was incorporated at its 3' end. For ligation to the binary target vector pZFNnptII (Figure 2), two HindIII restriction interfaces were adjacent to the expression cassette, followed by ligation to pZFNnptII_LbCpf1. The success of the insertion of the PcUbi::Cpf1::TPea expression cassette was verified by sequencing, and the binding regions of the primers used for sequencing were located both within the adjacent vector region and within the expression cassette (see Table C).

[0307] [Table 5]

[0308] After transcription into plant cells, the crRNA needs to be excised by two adjacent ribozymes. For this purpose, the coding sequences of the hammerhead ribozyme and the HDV ribozyme were placed adjacent to the precursor crRNA (Tang, X., LG Lowder, T. Zhang, AA Malzahn, X. Zheng, DF Voytas, Z. Zhong, Y. Chen, Q. Ren, Q. Li, ER Kirkland, Y. Zhang, and Y. Qi (2017), “A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants.” Nat Plants 3: 17018).

[0309] For complete ligation of individual protospacers in the coding sequence of the crRNA repeat, two BbsI detection sequences were incorporated between the crRNA repeat and the HDV ribozyme, and the overhangs used for cloning were adapted accordingly. To ensure identical expression intensities for cpf1 and crRNA, the crRNA ribozyme cassette was faculted with the PcUbi promoter sequence at the 5' end and the 3A terminator sequence at the 3' end. For subsequent ligation to the target vector pZFNnptII_Cpf1, two PstI interfaces were faculted onto the crRNA expression cassette and aligned as a synthetic DNA fragment. Protospacers were synthesized as complementary oligonucleotides and annealed according to a standard protocol. The resulting 24 bp DNA fragment was faculted with a 4 nt overhang relevant to ligation (see Table D).

[0310] [Table 6]

[0311] The efficiency of four crRNAs was tested in B. vulgaris leaves by Agrobacterium-mediated gene transfer. To confirm transformation efficiency, the pZFNtDTnptII plasmid was used for co-transformation. Transformation of leaf explants was performed by vacuum infiltration according to a standard protocol. After 6 days, tDT fluorescence was confirmed by fluorescence microscopy, and leaf explants with heterogeneous fluorescence were discarded. Ten days after infiltration, the leaf explants were rapidly frozen in liquid nitrogen, ground up, and genomic DNA was isolated by CTAB (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 NGS using the frequency of insertion edits (e.g., insertions, deletions, or base exchanges) compared to unedited sequences in genomic DNA, provided by an external service provider.

[0312] As a synthetic DNA construct, the most efficient crRNAs, 5'crRNA#3 and 3'crRNA#1, were aligned as a reversed expression cassette along with the aforementioned ribozyme, promoter, and terminator sequences. Two PstI restriction interfaces were placed adjacent to each other across the entire DNA construct for cloning into the target vector pZFNnptII_LbCpf1. After crRNA insertion, the LbCpf1 and crRNA expression cassette were ligated from the vector pZFNnptII_LbCpf1_crRNA to the pUbitDTnptII vector using HindIII.

[0313] To serve as a repair template for integration into the B. vulgaris genome via homologous recombination, a resistance gene expression cassette was fitted with 5' crRNA#3 at its 5' end and 3' crRNA#1 binding sequence at its 3' end. This allowed the resistance gene expression cassette to be excised from the plasmid using Cpf1. The entire DNA template was synthesized as an 87326 bp synthetic DNA fragment (SEQ ID NO: 80) and used directly as the vector backbone for transformation. The resistance gene plasmid and the pUbitDTnptII_LbCpf1_crRNA plasmid were introduced into B. vulgaris callus cultures using a gene cannon.

[0314] Transformation efficiency was determined by fluorescence microscopy using transient tDT fluorescence the day after transformation. Callus cultures were grown in shoot induction medium without selective pressure (kanamycin-free), and the regenerated shoots were subsequently examined for site-specific integration of the resistance-constituting resistance gene cassette. For this purpose, genomic DNA was isolated by CTAB. Integration of the resistance-constituting 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 with both primers. Shoots in which successful insertion of the expression cassette was verified in this way were identified by the following analysis of the resistance gene integration site. To verify insertion into the desired target sequence in the genome, the adjacent region of the resistance gene expression cassette was amplified by PCR. Primer binding was performed within the resistance gene DNA sequence, and the second primer binding was performed outside the 5'- or 3'-flanking homologous region of the inserted expression cassette (see Table E). The amplified DNA sequence was sequenced using the same primers to confirm 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) to genome-synchronized regions, all primer sequences were pre-compared to the B. vulgaris genome. For primer pCRBM_F3 (SEQ ID NO: 51), it was not possible to select a nucleotide sequence that would exclude binding to the wild-type sequence. Therefore, the 3'-flanking region was amplified in all shoots tested positive for the resistance gene, and site-specific insertions were verified by subsequent sequencing. As a result, the generated 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 with a third primer (pCRBM_S2, pCRBM_S3; see Table E) that had a binding site within the amplified sequence.To rule out nonspecific 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 to the internal reference genome of B. vulgaris. The primers were further tested by PCR for binding to the genome sequence of wild-type B. vulgaris plants.

[0315] To exclude the integration of resistance genes in other regions of the genome, targeted amplification was performed at target sites (target locus amplification, TLA).

[0316] [Table 7]

[0317] In addition to verifying and successfully inserting resistance gene expression cassettes into the B. vulgaris genome, we also confirmed the undesirable integration of plasmid DNA. For this purpose, the presence of plasmid DNA was confirmed by PCR in genomic DNA where successful insertion of resistance genes at the desired target site had already been verified. This allowed for amplification and subsequent sequencing of cpf1, crRNA ribozyme cassettes, and tDT sequences using the primers listed in Table F.

[0318] [Table 8]

[0319] Example 2: Introduction of a resistance-constituting gene as a transgene by gene transformation in Beta vulgaris subsp. vulgaris Transgenic approaches to creating circospora-resistant plants not only serve as a useful alternative to LRR genes as resistance-constituting genes, but also function as a means of inducing transgenic resistance events that confer novel circospora resistance or improve existing circospora resistance.

[0320] The binary vector pZFN-nptII-LRR was generated using the following standard cloning procedure: The cDNA of the resistance gene, as defined by Sequence ID No. 2, was cloned into the T-DNA of this vector, along with its native promoter sequence. The T-DNA further contained the neomycin phosphotransferase II (nptII) gene, which confers resistance to a wide range of aminoglycoside antibiotics, such as kanamycin or paromomycin. These antibiotic resistances were used for the selection of transgenic plant cells and tissues. The NOS promoter and pAG7 terminator were flanked by the nptII gene. The binary vector backbone further contained colE1 and pVS1 origins 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.

[0321] Sugar beet transformation was carried out according to Lindsey & Gallois (1990), “Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens.” Journal of experimental botany 41.5, 529-536.). For this purpose, “micropropagation shoots” of genotype 04E05B1DH5, which do not possess the resistance gene according to the present invention, were used as the starting material. The shoots were grown in the corresponding medium according to Lindsey & Gallois (1990). To induce as many meristematic tissues 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 containing the vector pZFN-nptII-LRR (Figure 3) was cultured in additional medium (see Lindsey & Gallois (1990)), and the corresponding antibiotic was further supplied for selection. Sections of meristematic tissue from the target shoots were incubated with Agrobacterium in supplemental medium for several hours (see Lindsey & Gallois (1990)). Plant explants and Agrobacterium were co-cultured in medium in the dark for at least two days (see Lindsey & Gallois (1990)), and the inoculated explants were subsequently incubated in supplemental medium in the dark for approximately two weeks (see Lindsey & Gallois (1990)). The explants were then further grown in supplemental medium (see Lindsey & Gallois (1990)) and subcultured to allow for the selection of transgenic tissues. To complete the selection stage and reduce the degree of chimera formation, the green "shoots" were transferred to medium H and all were grown for two weeks. Leaf material was then extracted from the green growing "shoots" and examined for the presence of transgenes by PCR. Suitable "shoots" were rooted in culture medium I and then transferred to a greenhouse for the creation of T1 seed stocks. Furthermore, the expression of transformation resistance genes was analyzed using leaf material obtained from these "shoots".

[0322] Analysis of expression levels RNA was isolated in vitro from the leaves of "shoots" and used in qRT-PCR. qRT-PCR was performed according to Weltmeier et al. 2011 (see Background of the Invention). Measurement values ​​were normalized to the reference gene PLT3_075_F09 (see Weltmeier et al. 2011). Expression was determined using the following primer sequences: [Table 9]

[0323] Resistance testing in sugar beets after inoculation with Cercospora beticola under greenhouse conditions: Pure cultures of known highly pathogenic Cercospora beticola were grown on vegetable juice agar in petri dishes (9 cm in diameter) under near-ultraviolet (NUV) light at 20°C. After 14 days, the surface of the agar with fungal growth was immersed in 10 ml of sterile water per petri dish, and conidia and mycelial fragments were carefully scraped off using a target carrier. Plants were inoculated using an inoculation density of 20,000 conidia / mycelial fragments per ml with 0.1% TWEEN20 added. At the time of inoculation, the plants had been cultivated in a greenhouse for 8-9 weeks. The upper and lower surfaces of the leaves were treated with the inoculation material. The plants were then incubated at 25°C for 5-7 days at 18 hours / 6 hours light / dark and approximately 100% humidity. The first circospora symptoms in sugar beet leaves appeared after 12-14 hours. Individual plant symptoms were periodically evaluated using the evaluation score shown in Table 1A. The results are shown below.

[0324] [Table 10]

[0325] Results of transgenic validation of resistance genes according to the present invention (see Table G) Test group 1 is a negative control. Its genotype is the same as test groups 2-11, but it has not undergone transformation. Therefore, expression could not be detected. Test group 4 underwent transformation, but expression could not be detected. Test groups 2, 3, and 5-11 are transformants that possess the resistance gene according to the present invention only through transformation. Test group 12 is a breeding line containing a non-transgenic type of the resistance gene according to the present invention. Evaluation scores for all lines were established after inoculating plant material with Cercospora beticola as described above. Test group 12 shows the highest resistance, indicated by a final value of 5.19.

[0326] The transgenic strains were evaluated using the following table: [Table 11]

[0327] Table H shows the evaluation scores for the transgenic validation group only. First, the mean values ​​for all transgenic test groups (excluding group 4) are shown. Below that, the evaluation scores for only the transgenic lines that showed an expression level of at least 10 (groups 3, 8, 9, and 10; see Table G) are shown. Here, the final evaluation score is 5.91. This is significantly higher resistance than the negative control in group 1, which had an evaluation score of only 6.46 (minimum significance = 0.4; see Table G). The best transgenic test group (group 8) shows even better resistance, with an evaluation score of 5.48 (see Table G).

[0328] It is worth mentioning that the expression level of transgenic insertions may be influenced by the integration locus. Since the expression levels were measured in vitro, the actual expression levels under infection conditions may be higher, especially if the resistance gene is under the control of a pathogen-inducible promoter.

[0329] Statistical evaluation of transgenic validation results [Table 12]

[0330] Table I shows the statistical evaluation of the assessment scores included in Table G. Each letter represents the assignment to a statistical group. For example, after the final evaluation (15 dpi), it is clear that test group 8 (transgenic validation) is in the same cluster as test group 12 (resistance origin), but in a different cluster than test group 1 (negative control). According to this, test group 8 has a significant difference from test group 1, but not a significant difference from test group 12.

[0331] In addition, a box plot analysis was performed. The box plot can be seen in Figures 4-7.

[0332] Example 3: Preparation of a resistant sugar beet plant according to the present invention based on genetic material obtained from Beta vulgaris subsp. maritima The process described below was based on pooling wild beet material to generate a circospora resistance gene pool. The accessions of Beta vulgaris subsp. maritima, which were used as starting material for the breeding program, are listed in the table below.

[0333] [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4]

[0334] As is evident from Table 2, the obtained genetic material has shown heterogeneous levels of resistance to Circospora and varying degrees of resistance across studies. For example, the accession 'PI 120704' scored 1 in one study and 9 in another. Because this publicly available data appears unreliable, seed material from the accession was sown, and the resulting plants were phenotypically screened for Circospora resistance. Approximately 150 partially resistant plants were selected. However, because the resistance observed in each plant may be the result of numerous genes all contributing only slightly, opportunities to establish measurable resistance or identify a single gene suitable for increasing resistance levels were limited. We decided to cross the approximately 150 resistant plants using natural pollination conditions. This approach also allowed for recombination within the genetic material. Crossing and selection were repeated over several generations to improve resistance levels. The best offspring were cloned and prepared for a gene mapping approach. The resistance mapping described here was combined with intensive phenotyping. By establishing a population of over 4000 differentiated offspring and developing a special recombination screen, the target region was narrowed and further isolated through analysis of informative recombinants (genotype and phenotype) in a series of resistance tests. This gene mapping, along with the creation of physical maps using WHG sequencing ("whole-genome sequencing"), comparative BAC (Bac-by-Bac) sequencing, and bioinformatics analysis, identified three recombinant genotypes that confirmed the resistance gene (one with one recombinant in a neighboring gene, and the other with two recombinants in neighboring genes). Considering specific requirements, the inventors placed high-frequency repeat structures, including tandem repeats with particularly high sequence homology, in the target region, which made marker development and, consequently, the identification of informative recombinants extremely difficult. The following steps were particularly critical to the location of the resistance gene's genetic structure: - Development of markers s4p0264s01, s4p2271s01, sxh0678s01, s4p4293s01, s4p4295s01, and s4p4301s01 (see Table 1B). - Detailed mapping combined with intensive phenotyping. In greenhouse experiments, 90 to 180 offspring were used per plant, and phenotypic analysis was performed using intensive statistical methods (e.g., t-tests, power analysis, etc.). - Identification and sequencing of BAC clones from a BAC pool of resistance genotypes. - Sequence evaluation, as well as the comparison of sequences and proteins between RR (i.e., resistant) and ss (i.e., susceptible) genotypes; in this regard, clear aggregation of RR and ss sequence data was not always possible due to the complexity of the sequences.

[0335] Within the framework of the breeding program, resistance derived from the Beta vulgaris subsp. maritima was crossed with a superior sugar beet line. Through several backcrosses using marker-assisted selection, the resistance gene was successfully introduced into the established sugar beet germplasm. Surprisingly, no undesirable effects on sugar yield, etc., were observed. Subsequently, proof of concept regarding the resistance gene in sugar beet was established through transformation and the generation of sugar beets into which the resistance gene had been introduced (see above). Following the success of this proof of concept, the generated sugar beet germplasm containing the resistance gene could be used to generate circospora-resistant sugar beet varieties.

[0336] Example 4: Screening for initiation accession of identified resistance genes After the resistance genes were identified, genetic resources (accessions as shown in Table 2) were screened using markers to identify accessions containing the resistance genes. The number of plants analyzed per accession varied depending on seed availability. This is shown in the table below.

[0337] [Table 14-1] [Table 14-2]

[0338] Each given plant in each accession was screened using 572 SNP markers located at the 5' and 3' positions of the resistance gene. The large number of markers allowed for the acquisition of haplotype patterns. However, none of the accessions used as starting material exhibited the haplotype of the CRBM line possessing the identified resistance gene. Most similarities were found in accession 48819 (designation DEU001) / 3555 (IDBBNR) (see Tables 2 and 3). The following table shows an excerpt of the full marker analysis, including the 5' and 3' positions of the resistance gene.

[0339] [Table 15]

[0340] The results of marker analysis (exemplified by the data shown in Table 4) indicate that the resistance gene according to the present invention cannot be traced back to one of the accessions shown in Table 2. Significant differences were observed even in plants with accession 48819, which has the strongest marker overlap between the resistant line according to the present invention and the resistant line. It is noteworthy that while a deletion was detected in the resistant line, accession 48819 showed a deletion at the same location. This may indicate that a significant gene rearrangement occurred at this locus during the generation of the resistance gene according to the present invention. This hypothesis may also explain why the resistance gene could not be traced back to the starting material of the breeding program.

[0341] Example 5: Preparation of Circospora-resistant seed stock The resulting sugar beet germplasm containing the resistance gene (results of Example 3) can be used to produce circospora-resistant sugar beet varieties. For this purpose, the gene was transferred by crossing a DH parent line that had been crossed with a DH parent line derived from another hybrid breeding pool. A hybrid variety containing resistance to circospora according to the present invention was obtained as a result. The seeds of this variety were separated (singularized), washed, and polished. The seeds were then subjected to priming and pelletizing as described in European Patent Application Publication No. 2002702. The resulting seed stock was placed in a cardboard package with an intermediate layer as a steam barrier. The resulting seed stock was suitable for sowing, growing, harvesting, and subsequent industrial sugar production.

Claims

1. In a method for identifying plants that are resistant or tolerant to Circospora, (i) (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 3; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 2; (c) A nucleotide sequence containing the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 53; (d) A nucleotide sequence that hybridizes with the complementary sequence of the nucleotide sequence according to (a), (b), or (c) by repeated washing in 4 × SSC at 65°C and then in 0.1 × SSC at 65°C for a total of approximately 1 hour; (e) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 3; (f) A nucleotide sequence that is at least 90% identical to the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 2 A step of detecting the presence or absence of a nucleotide sequence selected from the group consisting of the following: (ii) the step of detecting in the plant or a part of the plant the presence or absence of a polypeptide encoded by the nucleotide sequence defined in step (i); and / or (iii) A step to detect at least one marker gene locus in the nucleotide sequence or cosegregation region defined in step (i). Includes, The co-separated region is a genomic region that co-separates from the circospora resistance conferred by the polypeptide or the nucleotide sequence, The aforementioned co-separation region includes marker s4p1395s01 according to sequence number 101 and marker s4p0421s01 according to sequence number 221, and adjacent to them A method characterized by the following features.

2. (iv) The method according to claim 1, further comprising the step of selecting a plant that is resistant or tolerant to the circospora.

3. The method according to claim 1 or 2, wherein the detection in step (i) or (iii) is based on at least one polymorphism or single nucleotide polymorphism.

4. 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 relative to the nucleotide sequence. b) The at least one polymorphism or single nucleotide polymorphism is located within the range of 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 from the nucleotide sequence. c) The at least one polymorphism or single nucleotide polymorphism is detectable in seeds deposited with the NCIMB in Aberdeen, UK, under access number NCIMB 43646, or d) The at least one polymorphism or single nucleotide polymorphism is part of the cosegregation region, The nucleotide sequence is the nucleotide sequence defined in step (i) of claim 1, The coseparation region is the coseparation region defined in step (iii) of claim 1. The method according to claim 3, further characterized by the fact that

5. The following steps (i) and / or (iii) can be terminated by detection: a) Steps to prepare a plant, the tissue of the plant, the seeds of the plant, or their cells, and b) A step of extracting DNA from the plant, the plant tissue, the plant seeds, or their cells. The method according to any one of claims 1 to 4, further comprising:

6. The method according to any one of claims 1 to 5, comprising the use of at least two oligonucleotides.

7. The method according to claim 6, wherein the oligonucleotide is suitable for use as a primer in PCR and comprises the marker s4p1395s01 according to SEQ ID NO: 101 and the marker s4p0421s01 according to SEQ ID NO: 221, and can hybridize to adjacent genomic segments.

8. The method according to any one of claims 1 to 7, wherein the plant is a plant of the genus Branthus or a plant of the genus Spinach.

9. The method according to any one of claims 1 to 8, wherein PCR is used in step (i) and / or (iii), the PCR uses two allele-specific forward primers, the detection uses fluorescence resonance energy transfer, and the presence, absence or type of fluorescence is determined by a sensor.

10. The method according to claim 9, wherein one common reverse primer is used.

11. In plants of the genera Betula or Spinach, containing nucleic acid molecules encoding a polypeptide that can confer resistance to Circospora in plants expressing the polypeptide, The nucleic acid molecule (a) A nucleotide sequence encoding a polypeptide having the amino acid sequence according to Sequence ID No. 3; (b) A nucleotide sequence containing the DNA sequence according to Sequence ID No. 2; (c) A nucleotide sequence containing the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 53; (d) Nucleotide sequences that hybridize with the complementary sequence of the nucleotide sequence according to (a), (b), or (c) by repeated washing in 4 × SSC at 65°C and then in 0.1 × SSC at 65°C for a total of approximately 1 hour; (e) A nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 3; (f) A nucleotide sequence that is at least 90% identical to the DNA sequence by SEQ ID NO: 1 or SEQ ID NO: 2 It includes a nucleotide sequence selected from the group consisting of, The plants of the genus Branthus or Spinach further include an endogenous allele encoding an epsp synthase according to Sequence ID No. 223, which has an amino acid different from proline at position 179. A plant characterized by the following features.

12. The plant according to claim 11, further characterized by comprising a feature or combination of features selected from the list consisting of being a hybrid plant and / or a doubled haploid plant, having dominant resistance to the circospora, having the nucleic acid molecule or nucleotide sequence included as a genetic transfer or being homozygous, and / or having resistance to glyphosate.

13. The plant storage organ or leaf according to claim 11 or 12.

14. In the pelletized plant seeds according to claim 11 or 12, Pelleted seeds characterized in that the pelletized seeds contain nucleic acid molecules and endogenous alleles.

15. The epsp synthase having an amino acid different from proline at position 179, i) Sequence of sequence number 223 ii) A sequence having an amino acid different from serine and proline at position 179, or iii) A sequence that has at least 90% identity with sequence i) or ii) throughout its entire length. A plant according to claim 11 or 12, a storage organ or leaf according to claim 13, or a pelletized seed according to claim 14, comprising an amino acid sequence selected from the above.

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