Genes derived from SPINACIA TETRANDRA that encode proteins conferring resistance to PERONOSPORA FARINOSA, and spinach plants containing these genes

Abstract

The present invention relates to genomic DNA of Spinacia tetrandra, comprising a first or second genomic DNA fragment encoding a protein conferring resistance to the plant pathogen Peronospora farinosa. The present invention further relates to spinach plants having resistance to the plant pathogen Peronospora farinosa, comprising the first or second genomic DNA fragment. The present invention further relates to methods for providing and identifying spinach plants having resistance to the plant pathogen Peronospora farinosa. The present invention also relates to the use of one or more nucleic acid sequences or amino acid sequences for providing or identifying plants having resistance to the plant pathogen Peronospora farinosa.

Description

Technical Field

[0001] Specification The present invention relates to genomic DNA of Spinacia tetrandra comprising a first or second genomic DNA fragment encoding a protein conferring resistance to the plant pathogen Peronospora farinosa. The present invention further relates to spinach plants having resistance to the plant pathogen Peronospora farinosa and comprising a first or second genomic DNA fragment. The present invention further relates to methods of providing and identifying spinach plants having resistance to the plant pathogen Peronospora farinosa. The present invention also relates to the use of one or more nucleic acid or amino acid sequences for providing or identifying plants having resistance to the plant pathogen Peronospora farinosa.

Background Art

[0002] Spinach is commercially grown worldwide for its attractive and nutritious leaves. In 2018, spinach production was nearly 26 million tons worldwide. Spinach (Spinacia oleracea or S. oleracea) is a member of the family Amaranthaceae, subfamily Chenopodioideae. Other well-known members of this family include quinoa and beet. The latter are very important cultivated plants in agriculture, examples being sugar beet, red cabbage, and turnip.

[0003] In terms of nutritional value, while providing a relatively small amount of calories (23 in 100 grams of cooked spinach), spinach is a rich source of vitamins A, B2 (or folic acid), B6, C, E, and K, and also magnesium, manganese, calcium, potassium, iron, and dietary fiber.

[0004] The flowering of spinach is induced by (long) day lengths and, under optimal conditions, spinach can reach up to four generations per year, with the life cycle from seed to new harvest being completed within three months. The bottleneck in spinach production can sometimes be caused by seed dormancy.

[0005] Spinach is anemophilous, and its pollen can reach far. When a strain converts from female or mixed flowering to (all) male flowering within one week, it is considered male. Female strains remain as such for at least three weeks without producing any pollen. Plants with female flowering periods and plants with male flowering periods can be used as pollinators to produce spinach hybrids. Before the female plants produce male flowers, all female flowers are pollinated by male plants. Seed setting occurs rapidly within three days, and then it takes approximately one month for the seeds to mature.

[0006] Under optimal conditions, commercially available elite spinach strains are grown and harvested within 25 days.

[0007] Breeding has led to spinach plants that grow rapidly without premature flowering. Older varieties tend to have thinner leaves and a more intense, slightly bitter taste. Newer varieties have broader leaves and a milder taste. Also, recent types tend to bolt less under warm conditions and thus do not flower prematurely and produce seeds.

[0008] Spinach is cultivated for its leaves. Commercially available spinach may have dark green, round leaves. Leaf morphology is interesting to spinach breeders. A significant share of the cultivated spinach market is early-harvested baby leaf spinach. For spinach growers, it is important for the leaves to stand straight, which facilitates easy harvesting, and dark green color is desirable.

[0009] Spinach originated in Central Asia but is now produced worldwide. The traditional regions where spinach was grown as a crop were Europe and North America, but currently, the largest volume of spinach is produced in China. Spinach is produced for the food processing industry (canned or frozen spinach), and especially for the fresh market where baby leaf spinach is in demand. Breeders develop lines with the most suitable characteristics for a location or purpose.

[0010] An important development in the production and sale of fresh spinach was the introduction of bagged spinach. For this application, the desired leaf morphology is found in varieties where the leaves are not overly tightly packed and are partially savoy.

[0011] The basic types of spinach currently on the market are as follows: - Savoy varieties with dark green, curly, and crinkled leaves (mainly for the fresh market), - Flat or smooth leaf spinach with broad, smooth leaves that can be easily cleaned. This variety is used for the industry (canned or frozen spinach, as well as processed foods and baby food), - Semi-savoy is an intermediate type of spinach that has a texture comparable to savoy varieties but is as easy to clean as smooth varieties. It is cultivated for both the fresh market and the industry. - Oriental varieties that are heat-tolerant, have long petioles, pointed-tip leaves, some side lobes, and an upright growth habit as a plant.

[0012] Most spinach is produced at high plant densities for fresh market production, which creates an ideal environment for disease occurrence. Additionally, consumers are increasingly seeking vegetables obtained without the use of pesticides, fungicides, and insecticides, and without chemical treatment of seeds, leading to an increasing demand for organic vegetables. The challenge here is that in the absence of pesticides, fungicides, and insecticides, spinach plants are susceptible to plant diseases. Therefore, there is a need in the art for spinach cultivars that incorporate resistance to pathogens, preferably those that are naturally encoded, into their genes.

[0013] The most common pathogens causing diseases in spinach are Peronospora, Fusarium, Stemphyllium, Colletotrichum, Cercospora, and cucumber mosaic virus. The main disease in spinach is downy mildew caused by the oomycete pathogen Peronospora farinosa or Peronospora effusa (also called P. farinosa f sp. spinaciae or abbreviated as Pfs). The short life cycle of Pfs results in rapid growth of the pathogen on susceptible cultivars. Initially, small pale yellow irregular spots appear on the upper surface of the leaves, and downy growth covered with purple hairs appears on the lower surface of the spots. Spores are produced on the leaves 9 - 12 days after the initial infection and spread by wind and water splash. Infected leaves are no longer attractive for consumption and are prone to other secondary (microbial) infections.

[0014] One way to combat downy mildew is to spray fungicides on the plants. This approach is highly undesirable due to its significant impact on the environment, and furthermore, it is costly and labor-intensive. Additionally, half of the spinach produced agriculturally is destined for the organic market, rendering the use of fungicides to combat downy mildew inappropriate.

[0015] Peronospora farinosa is a pathogen that quickly overcomes or breaks resistance. It has been observed that newly introduced resistance genes are circumvented by the pathogen within 2 - 3 years, necessitating a constant search to identify new sources of resistance. To date, 17 official species have been described by the International Working Group on Peronospora effusa / farinosa / Pfs (IWGP). Since only a limited set of Peronospora resistance (RPF) genes originating from S. oleacea have been identified, wild relatives of spinach are potentially interesting sources of novel and alternative RPF genes.

[0016] Effector-triggered immunity (ETI) is part of the plant immune system, and NB-LRR proteins form a class of proteins that can initiate ETI. NB-LRR proteins derive their name from a central nucleotide-binding domain and a C-terminal leucine-rich repeat domain. Many plant disease resistance proteins are NB-LRR proteins, which recognize pathogen effectors and activate host defenses. The C-terminal LRR domain of NB-LRR proteins is highly irregular, with various lengths and different numbers of LRR repeats. The LRR domain is involved in pathogen recognition, and mutations in the C-terminal half of the LRR domain have been shown to affect recognition specificity. The LRR domain contains patches with epitopes involved in effector binding. Recognition of pathogen effectors through the LRR domain transmits a signal to the rest of the protein. WO2018059651 and related patent documents disclose the WOLF gene, which encodes a protein of the CC-NB-LRR family that confers resistance to Peronospora farinosa in spinach plants.

[0017] Considering that a significant portion of spinach production is grown using organic farming methods, there is a high demand for spinach varieties that are resistant to all known races of Peronospora farinosa (currently Pfs1-19). However, in fields of spinach cultivars that are fully resistant, i.e., resistant to Pfs1-19, the problems faced by growers change from Peronospora farinosa to other pathogens that can affect spinach. An example of such a pathogen is the fungus Stemphylium, the causal agent of Stemphylium leaf spot disease. Two species of Stemphylium have been described as causing disease on spinach, including Stemphylium beticola (formerly Stemphylium botryosum) and Stemphylium vesicarium. In recent years, S. vesicarium has been the most prevalent of the two species.

[0018] Stemphylium vesicarium produces typical conidia, which germinate on the leaf surface and cause small necrotic lesions with brownish rings on spinach leaves. Leaf spot disease can significantly reduce the quality and yield of spinach, especially for the fresh market. Differences in cultivar response to S. vesicarium have been observed.

[0019] Another pathogen that affects spinach production is the virus that causes spinach blight, cucumber mosaic virus (CMV). CMV belongs to the family Bromoviridae and the genus Cucumovirus, and shows a wide host range of 1,200 plant species in over 100 plant families. Economically important crops that can be affected by CMV infection include cucurbits, pepper, lettuce, celery, tomato, and legumes. Resistance encoded by genes against CMV can prevent the spread of CMV, especially when the rotation of highly susceptible crops is a common practice. Symptoms in spinach include yellowing of leaves, distortion of crown leaves, curling of leaves, growth inhibition, and plant death. Leaves with yellowing are not suitable for sale for fresh market spinach. Therefore, genetic resistance to CMV is a welcome addition in disease-resilient spinach plants.

Prior Art Documents

Patent Documents

[0020]

Patent Document 1

Non-Patent Documents

[0021]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0022] As shown, there is a rapid adaptation of Peronospora farinosa to the resistance encoded by the gene. New Peronospora farinosa isolates constantly emerge that can bypass resistance by avoiding host recognition. To counter this rapid adaptation, breeders either search for new resistance genes, or combine or stack known resistances, i.e., introduce two or more different Peronospora farinosa resistance genes into one plant, increasing the likelihood that the plant can overcome Peronospora infection, or decreasing the probability that the currently recognized 19 pathogen species can overcome resistance. Thus, there is a need in the art for new and alternative genes encoding resistance to Peronospora farinosa that enable breeders to develop new spinach cultivars resistant to the plant pathogen Peronospora farinosa. Meeting the above need in the art is one object of the present invention among other objects.

Means for Solving the Problems

[0023] Among other objects, this object is met by the present invention outlined in the appended claims.

[0024] Specifically, according to a first aspect, among other objects, the above object is - a first genomic DNA fragment having the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least 90% identity to SEQ ID NO: 1, or - a second genomic DNA fragment having the nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence having at least 90% identity to SEQ ID NO: 2 in the genomic DNA of Spinacia tetrandra, wherein the first or second genomic DNA fragment contains a gene encoding a protein that confers resistance to the plant pathogen Peronospora farinosa, is met by providing a genome.

[0025] In the context of the present invention, the "spinach reference genome" or "the spinach reference genome" refers to the spinach genome published by Cai, X., Sun, X., Xu, C. et al., Genomic analyses provide insights into spinach domestication and the genetic basis of agronomic traits. Nat Commun. 12, 7246 (2021). https: / / doi.org / 10.1038 / s41467-021-27432-z. Based on this publicly available spinach genome, a person skilled in the art can easily identify the corresponding positions in any spinach genome, for example, by aligning the sequences of the listed genomic fragments either completely or partially.

[0026] More specifically, among other purposes, the above object is - a first genomic DNA fragment having the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity with SEQ ID NO: 1, or - a second genomic DNA fragment having the nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity with SEQ ID NO: 2 is satisfied by providing genomic DNA of Spinacia tetrandra comprising, wherein the first or second genomic DNA fragment comprises a gene encoding a protein that confers resistance to the plant pathogen Peronospora farinosa.

[0027] In the context of the present invention, sequence identity is understood as the identity of consecutive nucleic acid or amino acid sequences over the entire sequence, using generally known alignment tools such as BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0028] Furthermore, according to another aspect, among other purposes, the above purpose is - The first genomic DNA fragment contains a gene encoding a first resistance protein, the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5, which confers resistance to the plant pathogen Peronospora farinosa, or - The second genomic DNA fragment contains a gene encoding a second resistance protein, the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6, which confers resistance to the plant pathogen Peronospora farinosa, and / or - The second genomic DNA fragment contains a gene encoding a third resistance protein, the amino acid sequence of SEQ ID NO: 22 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22, which confers resistance to the plant pathogen Peronospora farinosa is fulfilled by providing the genomic DNA of Spinacia tetrandra containing the same.

[0029] Furthermore, according to another aspect, among other purposes, the above purpose is - The first genomic DNA fragment contains a first cDNA nucleic acid sequence of SEQ ID NO: 3 or a sequence having at least 90% identity, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity with SEQ ID NO: 3, or - The second genomic DNA fragment contains a second cDNA nucleic acid sequence of SEQ ID NO: 4 or a sequence having at least 90% identity, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity with SEQ ID NO: 4, and / or - The second genomic DNA fragment comprises a third cDNA nucleic acid sequence having at least 90% identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity, with SEQ ID NO: 21 or SEQ ID NO: 21. is satisfied by providing the genomic DNA of Spinacia tetrandra.

[0030] According to another aspect, among other objects, the above object is satisfied by providing a protein comprising the amino acid sequence of SEQ ID NO: 5, 6, or 22, or a protein comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity with SEQ ID NO: 5, 6, or 22.

[0031] According to yet another aspect, among other objects, the above object is satisfied by providing a cDNA comprising the nucleic acid sequence of SEQ ID NO: 3, 4, or 21, or a cDNA comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity with SEQ ID NO: 3, 4, or 21.

[0032] According to another embodiment, among other objects, the above object is to provide a spinach plant resistant to the plant pathogen Peronospora farinosa, - the first or second genomic DNA fragment as defined above, and / or - the first or second resistance protein and / or the third resistance protein as defined above, and / or - the first or second cDNA sequence and / or the third cDNA sequence as defined above is satisfied by providing a spinach plant comprising.

[0033] Alternatively, the present invention is a spinach plant resistant to the plant pathogen Peronospora farinosa, - The above-defined first and second genomic DNA fragments, and / or - The above-defined first and second resistance proteins and / or third resistance protein, and / or - The above-defined first and second cDNA sequences and / or third cDNA sequence relates to spinach plants comprising

[0034] According to a preferred embodiment, among other purposes, the above object is fulfilled by providing the above-defined spinach plant having resistance at least to plant pathogen Peronospora farinosa species Pfs10 - Pfs19.

[0035] The inventors surprisingly discovered that by introducing a genomic fragment of Spinacia tetrandra on chromosome 4, a genomic fragment that brings additional resistance can be introduced on chromosome 3. This opens up the possibility of introducing further resistance into plants.

[0036] According to an even more preferred embodiment, among other purposes, the above object is fulfilled by providing the above-defined spinach plant having further resistance to plant pathogens Stemphylium vesicarium and / or CMV.

[0037] According to another aspect, among other purposes, the above object is fulfilled by providing the above-defined spinach plant, wherein the resistance can be obtained from, or is derived from, deposit NCIMB43993 for the first Spinacia tetrandra genomic fragment and / or deposit NCIMB43994 for the second Spinacia tetrandra genomic fragment.

[0038] The seeds of the plants Spinacia oleracea 2130195, deposit NCIMB 43993 and Spinacia oleracea 2025195, deposit NCIMB 43994 according to the present invention were deposited with NCIMB Limited, Craibstone Estate, 35 Ferguson Building, Bucksburn, Aberdeen AB21 9YA, United Kingdom on June 13, 2022.

[0039] Spinacia tetrandra and Spinacia turkestanica are wild-type relatives of modern spinach. Morphologically, they resemble ancient spinach, Spinacia oleracea. Also, they are either male or female and have pointed leaves with sharp angles at the tips. However, in the seed industry, there are problems with using Spinacia tetrandra and materials derived therefrom. S. tetrandra produces large, clustered, sharp seeds. This is highly undesirable in seed production for two reasons: (i) It is difficult to obtain a uniform coating for the sharp seeds, and (ii) The sharp seeds are not compatible with automatic seed planters.

[0040] In the context of the present invention, sharp seeds are defined as non-round seeds that have spikes and tend to cluster. This is in contrast to the agriculturally elite S. oleracea, which mainly produces round or rounded seeds that do not have spikes and do not tend to form clusters. Therefore, there is a need in the art to provide spinach plants that produce non-sharp seeds within a seed lot.

[0041] Thus, according to a particularly preferred embodiment, the present invention provides a spinach plant as defined above that produces seeds containing at most 25%, preferably 20%, more preferably 10%, or even more preferably 5% sharp seeds.

[0042] According to another aspect, the present invention provides a method for providing a spinach plant resistant to the plant pathogen Peronospora farinosa, comprising: operatively linked to appropriate expression and translation sequences, - the first or second genomic DNA fragment as defined above, or - the first, second, and / or third cDNA sequences as defined above introducing into a spinach plant susceptible to the pathogen Peronospora farinosa, and relates to a method for providing a spinach plant.

[0043] More specifically, the method includes the use of Agrobacterium and / or CRISPR Cas.

[0044] Even more specifically, in the above method, the first and second genomic DNA fragments are obtainable from, obtainable, or derived from deposit numbers NCIMB43993 and NCIMB43994, respectively.

[0045] According to yet another aspect, the present invention provides - isolating or providing plant material of a spinach plant, and - detecting in the plant material - the first or second genomic DNA fragment as defined above and / or - the first, second, and / or third resistance proteins as defined above, and / or - the first, second, and / or third cDNA sequences as defined above and relates to a method for identifying a spinach plant resistant to Peronospora farinosa.

[0046] ​Furthermore, the present invention relates to the use of one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 21, and / or the use of one or more amino acid sequences selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 22 for identifying or providing a plant having resistance to the plant pathogen Peronospora farinosa, preferably species Pfs10 - Pfs19.

[0047] The present invention is further illustrated by the following examples and the accompanying drawings.

Brief Description of the Drawings

[0048]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0049] Example 1. Peronospora farinosa - Disease Test Resistance to Peronospora farinosa f. sp. spinaciae (synonym P. effusa / hereinafter Pfs in this specification) was tested in a qualitative disease assay. Briefly, 10 - 14 days after sowing untreated seeds in soil, at least 8 plants were inoculated with a spore suspension of a single Pfs species or isolate. Pfs was maintained using spores stored at - 20 °C for up to 1 year on living susceptible host plants such as Viroflay or Blight or on plant material. The inoculated plants were incubated under plastic, at high humidity (80 - 100%) and at temperatures in the range of 16 °C - 20 °C. After 24 hours, the plastic was removed and the plants were evaluated 9 - 12 days after inoculation. If sporulation was observed on the cotyledons or true leaves, the plant was considered susceptible, and if no sporulation was observed, the plant was considered resistant.

[0050] To confirm the species, the differential set described in Table 1 was included in each disease test under the same environmental conditions. This differential set of Pfs was developed by the International Working Group on Peronospora farinosa (IWGP) and can be found on the website of the International Seed Federation (ISF). This differential set consisting of spinach varieties and near - isogenic lines (NILs) was used to determine the Pfs species. In this table, "-" indicates resistance (no sporulation), "+" indicates susceptibility (sporulation), "(-)" indicates intermediate resistance (sparse sporulation at the tip of the cotyledon), and "n.t." indicates that the current strain was not tested. This differential set and seeds of the Pfs species can be obtained from Naktuinbouw (Private Bag 40, NL - 2370 AA, Roelofarendsveen, the Netherlands, naktuinbouw.com).

[0051]

Table 1

[0052]

Table 2

[0053] Example 2. Stenfilium-disease test. Resistance to Stenfilium besikariium was tested in a qualitative disease assay. Briefly, 7 - 10 days after sowing untreated seeds in soil, at least 20 plants per line were transplanted into pots. The plants were inoculated 14 - 20 days after sowing, when the first true leaves were fully expanded. S. besikariium was maintained at - 80 °C in glycerol and replicated on potato dextrose agar (PDA). Spores were harvested, counted, and a spore suspension with a concentration of 1 * 10 4 spores / mL was produced. The inoculated plants were incubated under plastic, at high humidity (80 - 100%) and a temperature in the range of 20 °C - 22 °C. After 24 hours, the plastic was removed and the plants were evaluated 4 days after inoculation. If leaf spot disease was observed on the true leaves, the plant was considered susceptible; if no leaf spot disease was observed, the plant was considered resistant.

[0054]

Table 3

[0055] Example 3. CMV-disease test. Resistance to CMV was tested in a qualitative disease assay. Briefly, 7 - 10 days after sowing untreated seeds in soil, at least 20 plants per line were transplanted into pots. The plants were inoculated 14 - 20 days after sowing, when the first true leaves were fully expanded. CMV was maintained at 4°C as freeze - dried spinach leaves. CMV was first mechanically inoculated into Nicotiana benthamiana and subsequently propagated on a susceptible spinach variety. Spinach plants were evaluated 10 days after inoculation. Plants with yellowing leaves were considered susceptible, while plants without yellowing leaves were considered resistant.

[0056]

Table 4

[0057] Example 4. Seeds with significantly less sharpness. Spinacia tetrandra has sharp seeds - Figure 1. This type of seed (sharp seeds) tends to form clusters, which is undesirable. Sharp seeds are problematic because it is difficult to obtain a uniform seed coat layer and it is difficult to work with sharp seeds in an automatic seed sowing machine.

[0058] S. tetrandra was crossed with S. oleracea and backcrossed three times with S. oleracea - Figure 2. Evaluation by visual inspection revealed that seed lots resulting from this cross showed seeds with 50% less sharpness compared to the Spinacia tetrandra starting material.

[0059] Plants of deposit NCIMB43994 - Figure 3. These plants have only relatively small introgression of S. tetrandra, and their genome is mainly S. oleracea. By visual inspection, only 5% of the seed lot had sharp seeds, and when the seeds were sharp, it was evaluated that they were significantly less sharp than the seeds of S. tetrandra (starting material). Plants of deposit NCIMB43993 showed results similar to those of NCIMB43994 with respect to seed morphology (results not shown).

[0060] Example 5. Genomic DNA sequences, cDNA, and proteins of Spinacia tetrandra. Based on QTL analysis, two S. tetrandra accessions conferring resistance to Peronospora farinosa were identified and sequenced by Illumina technology. The sequencing data was mapped against the S. oleracea reference genome and a new genome assembly was performed. Previous marker analysis led to the identification of a QTL that is located at (3468181..3487380) on chromosome 4 of the S. oleracea reference genome. Within the QTL range, very interesting genes encoding putative resistance proteins were observed.

[0061] Comparative genomics between the S. oleracea reference genome and two new S. tetrandra genomes led to the identification of one chromosomal region of each new S. tetrandra genome assembly. In the first S. tetrandra accession, the genomic fragment is SEQ ID NO: 1 of 13.5 kb, and in the second S. tetrandra accession, the genomic fragment is SEQ ID NO: 2 of 17.5 kb.

[0062] Using Softberry software, the first genomic fragment (nucleic acid sequence according to SEQ ID NO: 1) and the second genomic fragment (nucleic acid sequence according to SEQ ID NO: 2) were visualized. Smaller fragments were selected within these fragments, where potential gene locations were identified and visualized - Figure 4. For both fragments, the identified genes have a similar structure, i.e., seven exons each containing a start and stop codon.

[0063] Based on BlastN, genes encoding putative resistance proteins were identified on chromosomal fragments, and the coding sequences of the target genes were predicted using Augustus. For the first S. tetrandra accession, the cDNA is SEQ ID NO: 3 with 3693 bp, resulting in a protein sequence of 1230 amino acids (SEQ ID NO: 5). The cDNA of the second S. tetrandra accession is SEQ ID NO: 4 (3693 bp), resulting in a protein sequence of 1230 amino acids (SEQ ID NO: 6).

[0064] The coding sequences of the resistance genes of the present invention for both S. tetrandra accessions show 97.3% and 97.3% homology, respectively, to the S. oleacea reference genome. The alignment indicates that the putative resistance protein underwent positive selection in the S. tetrandra resistance accession, resulting in Peronospora farinosa resistance. The protein sequences of the putative resistance genes for both S. tetrandra accessions show 95.1% and 95.2% homology, respectively, to the S. oleacea reference genome.

[0065] To confirm the prediction of the gene sequence of the second S. tetrandra accession (i.e., SEQ ID NO: 4, cDNA and SEQ ID NO: 6, protein), an RT-PCR reaction was performed.

[0066] RNA was isolated using the innuPREP Plant RNA Kit (Analytik Jena), and then cDNA was synthesized using the First strand cDNA Synthesis Kit (NEB). Primer pairs were designed for the predicted CDS in Augustus, and after the PCR reaction, this resulted in two distinct bands on the gel, suggesting the presence of two splicing variants. To confirm this result, the PCR product was sequenced using nanopore sequencing, which yielded the sequence of the first splice variant. In Figure 5, the predicted cDNA sequence, SEQ ID NO: 4 (green), is compared with the sequencing result obtained for splicing variant 1, SEQ ID NO: 21 (yellow), and both originate from the second S. tetrandra deposit. It can be observed that exon 1 differs between the prediction (Augustus) and splicing variant 1. In the case of splicing variant 1, exon 1 is longer at 2857 bp. The other exons were correctly predicted and their lengths are the same between the prediction (Augustus) and splicing variant 1.

[0067] The cDNA sequence of splicing variant 1 originating from the second S. tetrandra deposit is designated as SEQ ID NO: 21. The protein, which is the translation of the said cDNA, is designated as SEQ ID NO: 22 (originating from the second S. tetrandra deposit).

[0068]

Table 5

[0069] Example 6. LRR domain. The resistant protein sequences of the present invention that provide proteins derived from the reference genome, namely SEQ ID NO: 5 and SEQ ID NO: 6, contain the same protein domains. The lengths and orders of the domains are conserved among the proteins of the reference genome. However, in SEQ ID NO: 5 and SEQ ID NO: 6, several amino acid substitutions have occurred. Those skilled in the art are proficient in the methods for calculating sequence similarity and sequence identity. The sequence similarity of the amino acid sequences is calculated using the EBLOSUM62 matrix with the settings of gap open penalty: 12 and gap extension penalty: 2 using EMBOSS stretcher 6.6.0 (www.ebi.ac.uk / Tools / psa / emboss_stretcher).

[0070] It has previously been shown that the LRR protein domain plays an important role in obtaining resistance to pathogens. By comparing the LRR protein domains of the reference, SEQ ID NO: 5, and SEQ ID NO: 6 proteins, # identity: 316 / 333 (94.9%), # similarity: 321 / 333 (96.4%), # gap: 0 / 333 (0.0%) were obtained. Specific amino acid changes in the LRR domain are shown in Table 3 (Table 5).

[0071] Comparative genomics of nanopore-sequenced unit replication sequences using genes and splice variants predicted by Augustus showed that the LRR domain still exists in both versions.

[0072]

Table 6

[0073] Example 7. Introduction of a gene fragment conferring Peronospora farinosa resistance from Spinacia tetrandra into Spinacia oleracea using Agrobacterium tumefaciens. Transient transformation of plants using Agrobacterium tumefaciens with a resistance gene results in plants that are resistant to pathogens. To achieve this goal, molecular biology techniques can be used to design and obtain constructs that carry the resistance encoded by the genes according to the present invention. These constructs will carry the following: (1) A gene fragment encoding resistance from the S. tetrandra accession, designated as SEQ ID NO: 3, (2) A gene fragment encoding resistance from the S. tetrandra accession, designated as SEQ ID NO: 4.

[0074] Susceptible S. oleacea plants can be transformed using construct (1) or construct (2) using co - culture with A. tumefaciens. Additionally, positive and negative controls are included. When the transformation is complete, the transformants can be subjected to disease tests using P. farinosa isolates. It is predicted that the transformants will be resistant to P. farinosa infection while the wild - type plants will remain susceptible.

[0075] Example 8. Virus - induced gene silencing experiment (VIGS) to silence the resistance encoded by the gene from Spinacia tetrandra. VIGS vectors derived from Tobacco rattle virus (TRV) have been extensively described for studying gene function in Arabidopsis thaliana, Nicotiana benthamiana, Lycopersicon esculentum, and other plants. Prior to the experiment, the inventors received plasmids 0155 - 157 pTRV1 and 0158 - 160 pTRV2 - MCS from the Arabidopsis Biological Resource Center.

[0076] A genomic fragment from S. tetrandra, namely, (i) A gene fragment encoding resistance from the S. tetrandra deposit, designated SEQ ID NO: 3, (ii) A gene fragment encoding resistance from the S. tetrandra deposit, designated SEQ ID NO: 4 (However, these fragments are present in the S. tetrandra plants according to the invention), VIGS experiments can be carried out to confirm that they are responsible for the observed resistance phenotypes.

[0077] For this purpose, VIGS constructs targeting the previously described genomic fragments (SEQ ID NO: 3 and SEQ ID NO: 4) were designed using pssRNAit. Furthermore, a positive VIGS control was used, which targets the (phytoene desaturase) PDS gene. The negative control is an empty vector.

[0078] Due to the very high sequence identity between SEQ ID NO: 3 and SEQ ID NO: 4, the same siRNA molecules can be used. Three sequences were targeted (VIGS1, VIGS2, VIGS3).

[0079] For the PDS positive control, two sequences, PDS1 and PDS2, were targeted. See below.

[0080] [Table 7]

[0081] [Table 8]

[0082] Subsequently, these fragments were obtained using molecular biology techniques, cloned into the above VIGS plasmid, and transformed into Agrobacterium tumefaciens. Subsequently, co-cultivation of A. tumefaciens and the S. tetrandra plants according to the invention was carried out using the following specified ones.

[0083] [Table 9]

[0084] After VIGS silencing, the resulting transformed plants can be subjected to a disease test using P. farinosa species 17. Plants that were not transformed, while still having resistance to the pathogen, are expected to be more susceptible to P. farinosa in plants where the VIGS experiment was successful.

[0085] Furthermore, longer target sequences were designed and used. The longer fragments result in a number of small RNAs, such that the changes in successful silencing are significantly higher than when using only one small RNA as input. SEQ ID NO: 19 targets the resistance gene, the first and / or second genomic DNA from S. tetrandra. SEQ ID NO: 20 targets phytoene desaturase (control).

[0086]

Table 10

Claims

1. - A first genomic DNA fragment having a nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least 90% identity to SEQ ID NO: 1, or - A second genomic DNA fragment having a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence having at least 90% identity to SEQ ID NO: 2, comprising a genomic DNA of Spinacia tetrandra, wherein the first and second genomic DNA fragments contain a gene encoding a protein that confers resistance to the plant pathogen Peronospora farinosa, the genomic DNA.

2. - The first genomic DNA fragment contains a gene encoding a first resistance protein that confers resistance to the plant pathogen Peronospora farinosa, and the first resistance protein contains an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 85% identity to SEQ ID NO: 5, or - The second genomic DNA fragment contains a gene encoding a second resistance protein that confers resistance to the plant pathogen Peronospora farinosa, and the second resistance protein contains an amino acid sequence of SEQ ID NO: 6 and / or SEQ ID NO: 22, or a sequence having at least 85% identity to SEQ ID NO: 6 and / or SEQ ID NO: 22, The genomic DNA of Spinacia tetrandra according to Claim 1.

3. - The first genomic DNA fragment contains a nucleic acid sequence of SEQ ID NO: 3 or a sequence having at least 90% identity to SEQ ID NO: 3, or - The second genomic DNA fragment contains a nucleic acid sequence of SEQ ID NO: 4 and / or SEQ ID NO: 21, or a sequence having at least 90% identity to SEQ ID NO: 4 and / or SEQ ID NO: 21, The genomic DNA of Spinacia tetrandra according to Claim 1 or 2.

4. A protein containing the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

22.

5. A cDNA containing the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

21.

6. A spinach plant having resistance to the plant pathogen Peronospora farinosa, - The first and / or second genomic DNA fragments according to any one of Claims 1 to 3, and / or - One or more resistance proteins according to Claim 4, or - One or more cDNA sequences according to Claim 5 comprising a spinach plant.

7. The spinach plant according to claim 6, wherein the plant is resistant to at least the plant pathogen Peronospora farinosa species Pfs10 to Pfs19.

8. The spinach plant according to claim 6 or 7, wherein the plant is further resistant to the plant pathogens Stemphylium vesicarium and / or CMV.

9. The resistance is derived from, obtainable from, or is from the genomic fragment of the first Spinacia tetrandra with deposit NCIMB43993, or derived from, obtainable from, or is from the genomic fragment of the second Spinacia tetrandra with deposit NCIMB43994 The spinach plant according to any one of claims 6 to 8.

10. The spinach plant according to any one of claims 6 to 9, wherein the plant produces seeds containing up to 25%, preferably 20%, more preferably 10%, or even more preferably 5% sharp seeds.

11. - The first and / or second genomic DNA fragment according to any one of claims 1 to 3, and / or - One or more resistance proteins according to claim 4, or - One or more cDNA sequences according to claim 5 Seeds or plant parts produced by the spinach plant according to any one of claims 6 to 10, comprising.

12. A resistance gene conferring resistance to the plant pathogen Peronospora farinosa, wherein the gene encodes a protein having at least 85% sequence identity with SEQ ID NO: 5, SEQ ID NO: 6, and / or SEQ ID NO:

22.

13. A resistance gene conferring resistance to the plant pathogen Peronospora farinosa, wherein the gene can be obtained from the first and / or second genomic DNA fragment according to any one of claims 1 to 3.

14. A method for providing a spinach plant resistant to the plant pathogen Peronospora farinosa, the method comprising - The first or second genomic DNA fragment according to any one of claims 1 to 3, or - One or more cDNA sequences according to claim 5 operably linked to appropriate expression and translation sequences Introducing into a spinach plant susceptible to the pathogen Peronospora farinosa.

15. A method for providing a spinach plant according to claim 14, wherein the method includes a transformation step using Agrobacterium and / or CRISPR Cas.

16. The method according to claim 14 or claim 15, wherein the first or second genomic DNA fragment can be obtained from, is obtained from, or is derived from deposit numbers NCIMB43993 and NCIMB43994, respectively.

17. A method for identifying a spinach plant resistant to Peronospora farinosa, the method comprising: - isolating or providing plant material of the spinach plant; - in the plant material, - the first and / or second genomic DNA fragment according to any one of claims 1 to 3, or - one or more resistance proteins according to claim 4, or - one or more cDNA sequences according to claim 5 detecting A method comprising.

18. Use of one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 21, and / or Use of one or more amino acid sequences selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 22 for identifying or providing a plant resistant to the plant pathogen Peronospora farinosa, preferably species Pfs10 to Pfs19.

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