March gene contributing to downy mildew resistance

Abstract

The present invention relates to a modified MARCH gene, the wildtype of which comprises SEQ ID No. 1 and SEQ ID No. 3, or comprises a homologous sequence having at least 70% sequence identity to SEQ ID No. 1 and / or SEQ ID No. 3, which modified gene leads to resistance to downy mildew. The modification may leads to an altered gene expression, such as a reduced gene expression. In particular, the modified MARCH gene comprises a modification in SEQ ID No. 3, or in a homologous sequence having at least 70% sequence identity to SEQ ID No. 3. In one embodiment, the modification is on position 77 of SEQ ID No. 3, or on the corresponding position of a homologous sequence having at least 70% sequence identity. In one embodiment, the modified MARCH gene comprises SEQ ID No. 4. The invention further relates to a polynucleotide comprising a modified MARCH gene and a modified Malectin-like-RLK gene, preferably comprising SEQ ID No. 4 and SEQ ID No. 7. The invention further relates to a plant comprising a modified MARCH gene and to a marker for the identification of a modified MARCH gene.

Description

[0001] MARCH GENE CONTRIBUTING TO DOWNY MILDEW RESISTANCE

[0002] The present invention relates to a modified gene which contributes to resistance against downy mildew. The invention further relates to a plant comprising said modified gene, methods for producing such a plant, and methods for identification of the modified gene and selection of such a plant. The invention also relates to a marker for identification of the modified gene in a plant, and to use of said marker.

[0003] Downy mildew (DM) is one of the major problems that farmers encounter during the cultivation of many crops worldwide. The disease is well-known for affecting cucurbits in protected as well as in open-field cultivation. It is a mainly foliar disease that initially causes pale green to yellow angular spots on the upper side of the leaves. Once the disease progresses, the spots merge and turn chlorotic. The lesions appear angular because they are bound by leaf veins. Under certain environmental conditions, especially in wet and humid circumstances, inspection of the underside of the leaf reveals downy mycelial patches that come in a variety of colours. This downy material is the sporulation of the pathogen. Magnification of the sporulation will reveal the acutely and dichotomously branched sporangiophores bearing lemon-shaped sporangia. Advanced disease occurrence eventually results in necrosis and death of the leaves. Although the disease is almost exclusively restricted to the leaves, a severe disease incidence can result in significant losses in commercial agriculture due to a reduction of photosynthetic area that leads to stunted and weak plants, which in turn leads to a lower fruit production, and sunscalding of the fruit because of defoliation.

[0004] Downy mildew disease is caused by crop-specific obligate biotrophic oomycetes belonging to the family Peronosporaceae. In cucurbit crops such as cucumber, melon, squash, and watermelon the disease is caused by Pseudoperonospora cubensis [(Berk. And Curt.) Rost.]. The pathogens cannot survive on dead plants, nor can they be cultured on an artificial medium, but they are dependent on, or have to be maintained on, living plant material.

[0005] Control of downy mildew is generally done by using fungicides; it is a very serious and destructive disease if not treated. When circumstances are right, plants can be defoliated in a couple of days. The use of resistant varieties, optionally in combination with limited chemical control, is obviously a preferable option. A reduction in chemical use in agriculture is an ambition that helps in reducing both environmental and health concerns; in addition, protection based on a durable genetic resistance can last for a longer time, or even throughout the growing season. However, resistance to downy mildew has been researched for a long time, and it has been shown in various crops that the resistance is broken rather easily, especially when it is based on a single gene. In several cucurbit crops, such as cucumber, a high level of downy mildew resistance commonly requires a combination of QTLs or genes that each contribute to an increase in the resistance level. The requirement of multiple QTLs or genes makes the development of resistant varieties rather complicated and very time-consuming.

[0006] Sources of resistance are often still identified in genebank material, related wild species or subspecies, or non-cultivated landraces of a certain crop. However, the quantitative nature of the resistances that can be identified in this way for certain crops or diseases leads to complex breeding programmes, especially when only large QTL regions are found that cannot easily be followed through marker assisted breeding. In addition, the breeding material often suffers from pleiotropic effects that are caused by the introduction of unwanted characteristics of the non-agronomically elite or non-cultivated sources that are used, for example through linkage drag.

[0007] In cucumber, Cucumis sativus L., the search for DM resistance goes a long way back. A first DM resistant cucumber variety was released in the forties of the last century. This resistance was broken quite quickly, and new resistances were subsequently identified and commercialized. Because the pathogen appears to develop itself in sync with the resistances, the search for new resistant sources that can be used to stay ahead of the developing pathogen is continuously ongoing. In addition, combining multiple resistance genes for the same pathogen will usually lead to a more durable resistance that is less easily broken.

[0008] An overview of identified sources and causal genes for DM resistance in cucumber was published in 2020 Liu et al: Identification of novel loci and candidate genes for cucumber Downy Mildew resistance using GWAS. Plants 2020, 9, 1059). It shows that resistance QTLs or genes on all seven C. sativus chromosomes are identified and used in breeding programmes. However, the researchers of the present invention were not yet satisfied with the level of resistance that could be obtained by these published resources, and therefore set out to find new sources that would reveal other genes to further improve resistance to this important disease.

[0009] It is an object of the present invention to provide a gene that leads to resistance against downy mildew (DM).

[0010] Testing for downy mildew resistance is a continuous process in plant breeding of many crops. In the research that led to the present invention, advanced cucumber breeding lines with a complex pedigree developed from combinations of various accessions were tested for downy mildew in a bio-assay. Lines with an interesting level of resistance were identified, and a QTL study was done. This study located the resistance in 2 related lines, SOURCE-TO and SOURCE-M4, on chromosome 4 of C. sativus (Example 1). The pedigrees of both lines were not related to any of the sources that are described in the overview in Liu et A ‘ . (supra).

[0011] To make breeding with the resistance as efficient as possible, identification of the causal gene in the QTL region was performed. The observed variation in the DNA sequence of the fine-mapped region could not easily be correlated to variation in a gene as annotated in the public C. sativus genome, Cs9930_v3. Even though several downy mildew resistance conferring genes on chromosome 4 are known, none of the genes mentioned in Liu et al. supra) could be correlated to the cause of the downy mildew resistance in this new source. All observed in-gene variations in the fine-mapped QTL region were substitutions located in introns, and they did not lead to missense SNPs, i.e., they did not directly lead to changes in the encoded protein. Two particular SNPs in the fine-mapped region appeared to correlate really well with resistance. One of those SNPs was not present within the coding sequence of an annotated gene in the public genome, but in a 3’UTR region. The other SNP was present in an intron region. Table 3 shows the observed variations in the fine-mapped region.

[0012] The present invention provides a modified Membrane associated RCH-ring finger amino acid (AA) permease (MARCH) gene. The modified MARCH gene leads to downy mildew resistance. The wildtype of the MARCH gene comprises a coding sequence (CDS) according to SEQ ID No. 1, or comprises a sequence homologous thereto having at least 70% sequence identity to SEQ ID No. 1. The MARCH gene further comprises a wildtype 3’UTR sequence comprising SEQ ID No. 3, or comprises a homologous 3’UTR sequence having at least 70% sequence identity to SEQ ID No. 3. The invention thus provides a modified MARCH gene, comprising a CDS and a 3’UTR, wherein the wildtype of the CDS comprises SEQ ID No. 1 or a homologous sequence having at least 70% sequence identity to SEQ ID No. 1 and the wildtype of the 3’UTR comprises SEQ ID No. 3, or a homologous sequence having at least 70% sequence identity to SEQ ID No. 3, which modified gene leads to resistance to downy mildew.

[0013] Further analysis of the region with the SNP in the 3’UTR sequence was done. Blasting of the sequence in the NCBI database resulted in a hit with NCBI_LOC101209559, which was termed to be an ‘uncharacterized protein’. Additional study on the gene showed the presence of several potentially interesting conserved domains in the encoded protein, such as a RING- variant domain and a RING_CH-C4HC3_MARCH domain, which are present in an overlapping region of the protein. Both are domains involved in membrane-associated processes. Also, an Amino Acid Permease domain was observed to be present, which is involved in amino acid transport. These findings, which were determined during the internal research, resulted in the conclusion that the uncharacterized protein was to be designated as a Membrane associated RCH- ring finger AApermease (MARCH) protein. The wildtype of this protein is presented as SEQ ID No. 2. This determination was very surprising, since MARCH genes were thus far not known for their involvement in disease resistance in plants.

[0014] In one embodiment, the modified MARCH gene comprises a modification that leads to an altered gene expression. The modification in the MARCH gene that leads to downy mildew resistance is preferably a modification that affects gene expression, such as a modification that affects the transcription process, or a modification that affects the translation process. In one embodiment the altered gene expression is a reduced gene expression.

[0015] In one embodiment the modified MARCH gene comprises a modification in the 3’UTR region of the gene, which modification leads to downy mildew resistance. The wildtype 3’UTR region comprises SEQ ID No. 3. The 3’-UTR region, which immediately follows the CDS, can harbour cis-acting elements, which are often, although not solely, involved in the regulation of post-transcriptional gene expression. Cis-regulatory elements include binding sites for transcription factors (‘TF binding sites’), both for transcriptional repressors as well as activators, and microRNA target sites. The modification in the 3’UTR region can lead to an altered gene expression through a change in polyadenylation of the mRNA, a change in translation efficiency of the mRNA, a change in localization of the mRNA, a change in stability of the mRNA, a change in miRNA binding to binding sites within the 3’UTR resulting in inhibition of translation, a change in miRNA binding to binding sites within the 3’UTR resulting in increased transcript degradation, a change in binding of repressor proteins to 3’UTR silencer regions resulting in inhibition of mRNA expression, or a combination of one or more of these changes. The modification in the 3’UTR region is in particular a modification that alters the functioning of the 3’UTR.

[0016] In one embodiment, the modified MARCH gene comprises a C to T modification on position 77 of SEQ ID No. 3, or on the corresponding position of a homologous sequence, which leads to a modified 3’UTR region comprising SEQ ID No. 4 or comprising a homologous sequence thereof. The modified MARCH gene of this embodiment thus comprises a 3’UTR comprising SEQ ID No. 4, or comprising a homologous 3’UTR sequence having at least 70% sequence identity to to SEQ ID No. 4 and having the modification on the corresponding position. Position 77 of SEQ ID No. 3 corresponds to position 22743655 of the public genome Cs9930_v3. This modification is abbreviated as a C77T SNP in relation to SEQ ID No. 3. The modified MARCH gene comprising the C77T SNP can be identified by a marker designed to identify a T on position 77 of SEQ ID No. 4. Such marker for example comprises a forward primer comprising SEQ ID No. 10 in combination with a reverse primer comprising SEQ ID No. 11.

[0017] As used herein, a wildtype MARCH gene is a gene comprising a CDS sequence comprising SEQ ID No. 1, or a homologous MARCH gene comprising a CDS sequence having at least 70% sequence identity to SEQ ID No. 1; or a gene encoding a MARCH protein comprising SEQ ID No. 2, or a gene encoding a homologous MARCH protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID No. 2. A wildtype MARCH gene further comprises a wildtype 3’UTR sequence comprising SEQ ID No. 3, or comprises a homologous 3’UTR sequence having at least 70% sequence identity to SEQ ID No. 3.

[0018] In one embodiment, the modification of a modified MARCH gene preferably comprises a T on position 77 of SEQ ID No. 4 and the homologous sequence having at least 70% sequence identity to SEQ ID No. 4 therefore retains at least a T on position 77 of SEQ ID No. 4, or on the corresponding position of the homologous sequence.

[0019] Further embodiments comprise the MARCH gene, the 3’UTR according to SEQ ID No. 3 of which comprises one or more other modifications leading to DM resistance. A sequence that is homologous to the 3’UTR sequence comprising SEQ ID No. 3 with a modification is a sequence having at least 70% sequence identity to SEQ ID No. 3, and having the modification.

[0020] As used herein, sequence identity is the percentage of nucleotides or amino acids that is identical between two sequences after proper alignment of those sequences. The person skilled in the art is aware of how to align sequences, for example by using a sequence alignment tool such as BLAST®, which can be used for both nucleotide sequences and protein sequences. To obtain the most significant result, the best possible alignment that gives the highest sequence identity score should be obtained. The percentage sequence identity is calculated through comparison over the length of the shortest sequence in the assessment, whereby in the present case a sequence represents a gene that at least comprises a start codon and a stop codon, or a complete protein encoded by such a gene.

[0021] As used herein, a sequence having at least 70% sequence identity relates to a sequence having in order of increased preference at least 70%, 75%, 77%, 80%, 83%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0022] As used herein, physical positions are based on the public Cucumis sativus genome of Cs9930, version 3 (v3), which is published in the NCBI database and can for example be found at https: / / www.ncbi.nlm.nih.gOv / datasets / genome / GCF_000004075.3 / .

[0023] An X000Y mutation, SNP, or substitution means that the wildtype sequence has a nucleotide or amino acid X on position 000, which is changed to nucleotide or amino acid Y in the modified sequence.

[0024] The modification in the MARCH gene of the invention leads to resistance against downy mildew in a plant comprising the modified MARCH gene, in particular resistance to Pseudoperonospora cubensis. The downy mildew resistance conferred by the modified MARCH gene on its own is a certain level of resistance. The presence of the modified MARCH gene in combination with other downy mildew resistance genes leads to an improvement of the resistance conferred by the other genes. Both the inherent resistance of the MARCH gene itself and the improving effect the modified MARCH gene has on the resistance conferred by other downy mildew resistance genes that are present in the same genome will be called “resistance” herein.

[0025] In one embodiment, the modification that leads to resistance against downy mildew, in particular to Pseudoperonospora cubensis, is a modification in the 3’UTR of the MARCH gene. Said modification comprises in particular a C to T modification on position 77 of SEQ ID No. 3, which leads to SEQ ID No. 4. Resistance to downy mildew, as used herein, is determined by a bio-assay using a susceptible (S) downy mildew control for comparison, and for determining that the assay is carried out correctly. A difference in disease occurrence determined in a properly designed assay, as is known to the skilled person, defines resistance. A suitable susceptible control is for example the publicly available variety Liszt Fl. Resistance caused by the modified MARCH gene in a certain plant or line can also be determined by comparing with an isogenic plant or line that lacks the modified MARCH gene.

[0026] To determine resistance a bio-assay is used in which seeds of the accessions to be tested are sown in seedling trays and seedlings are transplanted at around 1 week after sowing. Seedlings are transplanted into a field in a region for which it is known that downy mildew is occurring. Downy mildew can only be maintained on living plants and efficient inoculation can therefore be done through infected plants growing in the vicinity of the plant to be tested. Because downy mildew is a worldwide occurring disease, such regions with downy mildew are easily identified by the skilled person. Inoculation is then occurring by field inoculation, which is a continuous process once the disease enters the crop. Once the susceptible control, for example Liszt Fl, for which symptoms are known, starts to show clear symptoms, resistance scoring can start. Resistance is suitably scored on a scale of 1-9; a description of the scales of the scores 1-9 is presented in Table 1. The score on the scale of 1-9 is called herein the “phenotypic score”.

[0027] Depending on the disease incidence, start of the observation is usually at around 4 weeks after transplanting, and the susceptible control should then have a score of 5 or lower according to Table 1. Resistance is suitably scored 2 times within one week, for a minimum of 4 days, 7 days, 10 days, 12 days, 14 days, 18 days, or 21 days, to get a reliable impression. After 10 days, the susceptible control scores an average of between 2.0 and 3.0 according to Table 1, if there is a heavy disease incidence, or the susceptible control has an average phenotypic score that is reduced by at least 1.0 as compared to the start of the scoring, to determine the presence of resistance.

[0028] Resistance to downy mildew is defined as a higher phenotypic score in the above bio-assay when compared to a susceptible control and / or a higher phenotypic score in the above bio-assay when compared to an isogenic plant that does not have the modified MARCH gene of the present invention. Such isogenic plant optionally has one or more other downy mildew resistance conferring genes. A higher phenotypic score can be a higher score determined at a certain observation moment, for example at 4 weeks after transplanting in a field bio-assay, if the disease is present and the susceptible control has a score of 5 or lower on the scale of Table 1. Resistance can also be a more durable resistance which shows as maintaining a higher phenotypic score over a period of time, for example after assaying for at least 5 days, 7 days, 10 days, 12 days, 14 days, 18 days, 21 days, or for a longer period of time, in which period the phenotypic score is determined at least 2 times, preferably at least 3 times.

[0029] A plant of the present invention is a plant having a modified MARCH gene as defined herein. Determining whether a given plant is a plant of the invention is suitably done by detecting the presence of the modified MARCH gene as described herein in the genome of that plant.

[0030] The present invention relates to a plant comprising a modified MARCH gene of the invention. The plant comprising the modified MARCH gene is preferably a plant of the Cucurbitaceae family, in particular a plant of the species Cucumis sativus, Cucumis melo, Cucurbita pepo, Cucurbita maxima, Cucurbita moschata, or Citrullus lanatus. A plant of the invention is most preferably a plant of the species Cucumis sativus. A plant of the invention is preferably a cultivated plant, which is non-wild and has agronomical value, and is in particular agronomically elite.

[0031] During the research for the present invention, it was determined that the transfer of a modified MARCH gene from a resistant to a susceptible plant resulted in resistance to downy mildew, thereby confirming that the modified MARCH gene was the cause of the resistance, as described herein.

[0032] As used herein, a plant that has downy mildew resistance due to a modified MARCH gene has a phenotypic score that is significantly higher than the score of a susceptible control. A plant that has downy mildew resistance due to a modified MARCH gene alternatively has a phenotypic score that is significantly higher than the score of an isogenic plant lacking the modified MARCH gene, when scoring according to Table 1 is used.

[0033] In one embodiment, a plant of the invention is a Cucurbitaceae plant comprising a modified MARCH gene which comprises a modified 3’UTR according to SEQ ID No. 4, or which comprises a 3’UTR sequence that is homologous to SEQ ID No. 4. In a preferred embodiment said Cucurbitaceae plant is a Cucumis sativus plant.

[0034] In one embodiment, a plant of the invention is a Cucumis sativus plant comprising the modified MARCH gene as comprised in the genome of a C. sativus plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 44324.

[0035] In one embodiment, a plant of the invention is a Cucumis sativus plant comprising the modified 3’UTR of the MARCH gene as comprised in the genome of a C. sativus plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 44324.

[0036] In one embodiment, a plant of the invention is a C. sativus plant deposited as NCIMB 44324, or a progeny plant thereof that has retained the modified MARCH gene that is present in said deposit. In one embodiment, a plant of the invention is a C. sativus plant deposited as NCIMB 44324, or a progeny plant thereof that has retained the modified 3’UTR of the MARCH gene that is present in said deposit.

[0037] A plant of the invention comprises a modified MARCH gene homozygously or heterozygously, i.e. a modified MARCH gene can be present on both chromosomes of a chromosome pair in the genome of a plant, or on only one chromosome of a chromosome pair.

[0038] A plant of the invention comprises a plant of an inbred line, a hybrid (Fl) plant or variety, an open pollinated (OP) variety, a doubled haploid, or a plant of a segregating population.

[0039] The downy mildew resistance caused by the modified MARCH gene of the invention is semi-dominant which leads to an intermediate inheritance of the resistance in a plant of the invention. As used herein, intermediate means that a higher level of resistance is found when a modified MARCH gene of the invention is homozygously present. The heterozygous presence of a modified MARCH gene of the invention however already confers a certain level of downy mildew resistance. The downy mildew resistance of both homozygous and heterozygous plants makes the plants more suitable for cultivation under conditions where downy mildew is present. Therefore, both heterozygous and homozygous plants have an improved value in use for downy mildew resistance. In addition, heterozygous plants can be used for development of homozygous plants through crossing and selection, which heterozygous plants also for that reason form a part of this invention. In a preferred embodiment, the modified MARCH gene is homozygously present in a plant.

[0040] The modified MARCH gene of the invention can be combined with other genes or QTLs that confer downy mildew resistance, to obtain a higher, improved, and / or more durable level of resistance. A plant having an improved or more durable downy mildew resistance due to a modified MARCH gene is thus a plant comprising also other genes or QTLs that lead to downy mildew resistance, in addition to the modified MARCH gene.

[0041] In experiments done for the present invention, the modified MARCH gene was combined with a modified Malectin-like-RLK gene. The combination of both genes resulted in a higher level of downy mildew resistance than the presence of only one of said genes in C. sativus. The wildtype of the Malectin-like-RLK gene comprises SEQ ID No. 5, or comprises a homologous sequence having at least 70% sequence identity to SEQ ID No. 5. The wildtype Malectin-like-RLK protein encoded by SEQ ID No. 5 comprises SEQ ID No. 6.

[0042] In one embodiment, a modification in the Malectin-like-RLK gene conferring downy mildew resistance leads to a reduced expression or absence of expression, or the modification leads to a non-functional protein. A non-functional protein comprises a protein that does no longer function, or a protein that has a different function than its wildtype function. A nonfunctional protein can be caused by a mutation causing an early stop codon in the gene sequence. In one embodiment, the modified Malectin-like-RLK gene comprises SEQ ID No. 7. The modified Malectin-like-RLK gene comprising SEQ ID No. 7 has a modification in an intron that leads to an alternative splice site, in particular an earlier splice acceptor. The earlier splice acceptor results in a frame-shift, which in turn encodes a truncated protein comprising SEQ ID No. 8 because of a potential early stop codon. The modification in the intron comprises a C to G modification on position 2063 of SEQ ID No. 5, which leads to SEQ ID No. 7. The modified Malectin-like-RLK gene comprising SEQ ID No. 7 can be identified by a marker that is designed to identify the presence of a G on position 2063. Such marker for example comprises a forward primer comprising SEQ ID No. 13 in combination with a reverse primer comprising SEQ ID No. 14.

[0043] Since homologues of the MARCH gene and the Malectin-like-RLK gene are also present in other crops, especially in cucurbits, and downy mildew is known to infect many plant species, the finding of the involvement of these genes in downy mildew resistance can also be applied to downy mildew resistance improvement in other crops.

[0044] In one embodiment a downy mildew resistant plant of the invention, in particular a downy mildew resistant Cucumis sativus plant, comprises a modified MARCH gene and a modified Malectin-like-RLK gene. In a preferred embodiment, said C. sativus plant comprises a modified MARCH gene comprising SEQ ID No. 4 and a modified Malectin-like-RLK gene comprising SEQ ID No. 7.

[0045] The invention further relates to a polynucleotide or a nucleic acid molecule comprising a modified MARCH gene and a modified Malectin-like-RLK gene. Said polynucleotide or molecule in particular comprises SEQ ID No. 4 and SEQ ID No. 7. The invention also relates to a plant comprising said polynucleotide or nucleic acid molecule as part of the genomic DNA.

[0046] The modified MARCH gene of the invention, optionally in combination with a modified Malectin-like-RLK gene, can further be combined with any other downy mildew resistance conferring genes, for example the genes that are described in Liu et al. (supra), to obtain an even higher and / or more durable resistance. The modified MARCH gene and / or modified Malectin-like-RLK gene can in particular be combined with a modified CsAAP2 gene, that is also located on chromosome 4. This gene was identified as a causal gene for DM resistance in the QTL on chromosome 4 of the well-known downy mildew resistant source PI197088. WO2020239495 describes that a reduction or absence of CsAAP expression leads to resistance or reduced susceptibility to downy mildew in cucumber. In the research for the present invention, a modified CsAAP2 gene with a loss-of-function allele, which was an allele having a TE insertion in exon 4, as described in Examples 4 and 5 of WO2020239495, was combined with the modified MARCH gene and the modified Malectin-like-RLK gene. The combination of these 3 modified genes was tested during the research for the present invention, and the result is described in Example 6. It was determined that the combination of the 3 modified genes led to a more durable downy mildew resistance in C. sativus.

[0047] The invention further relates to a seed that comprises the modified MARCH gene of the invention, which seed can grow into a plant of the invention. The invention also relates to use of said seed for the production of a plant of the invention, by growing said seed into a plant. The invention also relates to a plant part of a plant of the invention, which comprises a fruit or a seed, wherein the plant part comprises a modified MARCH gene in its genome. In a preferred embodiment, the modified MARCH gene is homozygously present in a seed.

[0048] The invention further relates to a method for seed production, comprising growing a plant from a seed of the invention that comprises a modified MARCH gene of the invention, allowing the plant to produce a fruit with seed, harvesting the fruit, and extracting those seed. Production of the seed is suitably done by selfing or by crossing with another plant that is optionally also a plant of the invention. The seed that is so produced has the capability to grow into a plant that is resistant to downy mildew. The method in particular relates to production of Cucumis sativus seed. In one embodiment, at least one of the parent plants used in a crossing comprises the modified MARCH gene of the invention homozygously. In one embodiment both parent plants used in the crossing are a plant of the invention. In one embodiment both parent plants used in the crossing comprise the modified MARCH gene of the invention homozygously.

[0049] The invention further relates to hybrid seed and to a method for producing said hybrid seed, comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and / or the second parent plant is a plant of the invention comprising a modified MARCH gene of the invention homozygously. The hybrid seed is in particular seed of Cucumis sativus. The resulting hybrid plant that can be grown from the hybrid seed, comprising the MARCH gene of the invention, which hybrid plant has resistance to downy mildew, is also a plant of the invention.

[0050] In a preferred embodiment, the resulting hybrid plant is a Cucumis sativus plant.

[0051] In a preferred embodiment, the modified MARCH gene is homozygously present in both parent plants of the hybrid, in particular of the Cucumis sativus hybrid plant.

[0052] In a preferred embodiment a hybrid Cucumis sativus plant comprises a modified MARCH gene and a modified Malectin-like-RLK gene, more preferably at least one of said genes homozygously, most preferably both genes homozygously. The modified MARCH gene herein preferably comprises SEQ ID No. 4 and SEQ ID No. 1 , and the modified Malectin-like-RLK gene herein preferably comprises SEQ ID No. 7.

[0053] The present invention relates to a method for producing a plant that is resistant to downy mildew, comprising introducing a modification in a MARCH gene, which modification leads to downy mildew resistance. Said method comprises the introduction of a deletion, a substitution, or an insertion of one or more nucleotides in the coding sequence or the genomic sequence of a MARCH gene, or in the 3’UTR of a MARCH gene. The introduction of such a modification can be done by a mutagenesis approach using a chemical compound, such as ethyl methane sulphonate (EMS); or by using physical means, such as UV-irradiation, fast neutron exposure, or other irradiation techniques.

[0054] The invention relates in particular to a method for producing a Cucumis sativus plant that is resistant to downy mildew, comprising introducing a modification in a MARCH gene comprising SEQ ID No. 1 and / or SEQ ID 3, or in a homologous sequence having at least 70% sequence identity thereto. The method comprises in particular the creation of a mutation on position 77 in SEQ ID No. 3, preferably the modification from a C into a T on said position.

[0055] Introduction of a modification can also be done using a more specific, targeted approach including targeted genome editing by means of homologous recombination, oligonucleotide -based mutation introduction, zinc-finger nucleases (ZFN), transcription activatorlike effector nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems, which are known to the skilled person.

[0056] Introduction of a modified MARCH gene of the invention can also be done through introgression from a plant comprising said modified MARCH gene, for example from a plant that was deposited as NCIMB 44324, or from progeny thereof that has retained the modified MARCH gene, or from another plant that is resistant to downy mildew, and in which a modified MARCH gene, in particular the modified gene of the invention, was identified. Breeding methods such as crossing and selection, backcrossing, recombinant selection, or other breeding methods that result in the transfer of a genetic sequence from a resistant plant to another, optionally susceptible, plant can be used. A downy mildew resistant plant can be of the same species or of a different and / or wild, or non-cultivated, species. Difficulties in crossing between species can be overcome through techniques known in the art such as embryo rescue. Cis-genesis can also be applied to transfer a modified MARCH gene from one species to another. Progeny of the deposit NCIMB 44324 can be sexual or vegetative descendants of that deposit, which can be selfed and / or crossed, and can be of an Fl, F2, or further generation as long as the descendant of the deposit still comprises the modified MARCH gene of that deposit. A plant produced by such method is also a part of the invention. A plant used in this method and / or produced by this method is preferably a Cucumis sativus plant.

[0057] Transgenic techniques used for transferring sequences between plants that are sexually incompatible can also be used to produce a plant of the invention, by transferring a modified MARCH gene from one species to another. Techniques that can suitably be used comprise general plant transformation techniques known to the skilled person, such as the use of an Agrobacterium-mediated transformation method. The invention also relates to a method for the production of a plant which has resistance to downy mildew, said method comprising: a) crossing a plant comprising a modified MARCH gene of the invention with another plant; b) optionally performing one or more rounds of selfing and / or crossing a plant resulting from step a) to obtain a further generation population; c) selecting from the population resulting from the cross of step a), or from the further generation population of step b), a plant that comprises a modified MARCH gene as defined herein, which plant is resistant to downy mildew.

[0058] In a preferred embodiment, the above method relates to the production of a Cucumis sativus plant.

[0059] The invention also relates to a method for the production of a plant that has resistance to downy mildew, said method comprising: a) crossing a first parent plant comprising a modified MARCH gene of the invention with a second parent plant, which is another plant that does not comprise a modified MARCH gene of the invention; b) backcrossing the plant resulting from step a) with the second parent plant for at least three generations; c) selecting from the third or higher backcross population a plant that comprises at least the modified MARCH gene of the first parent plant of step a).

[0060] In a preferred embodiment, the above method relates to the production of a Cucumis sativus plant.

[0061] The invention additionally provides for a method of introducing another desired trait into a plant that is resistant to downy mildew due to a modified MARCH gene, comprising: a) crossing a plant comprising a modified MARCH gene of the invention with a second plant that comprises the other desired trait to produce Fl progeny; b) optionally selecting in the Fl for a plant that comprises the modified MARCH gene and the other desired trait; c) crossing the optionally selected Fl progeny with one of the parents for at least three generations, to produce backcross progeny; d) selecting backcross progeny comprising the modified MARCH gene and the other desired trait; and e) optionally repeating steps c) and d) one or more times in succession to produce selected fourth or higher backcross progeny that comprises the modified MARCH gene and the other desired trait. In a preferred embodiment, the above method relates to the introduction of another desired trait in a downy mildew resistant Cucumis sativus plant that comprises a modified MARCH gene.

[0062] Optionally, selfing steps are performed after any of the crossing or backcrossing steps in above described methods. Selection of a plant comprising the downy mildew resistance, from the modified MARCH gene, optionally in combination with the other desired trait, can alternatively be done following any crossing or selfing step of the method. The other desired trait can be selected from, but is not limited to, the following group: resistance to bacterial, fungal or viral diseases, insect or pest resistance, improved germination, plant size, plant type, vigour, improved shelf-life, improved yield, water stress and heat stress tolerance, and male sterility. The other desired trait can also be downy mildew resistance caused by a different gene than the MARCH gene. The invention includes a plant produced by any of these methods and a fruit obtained therefrom.

[0063] The invention further relates to a method for the production of a plant comprising a modified MARCH gene of the invention, in particular a MARCH gene with a modified 3’UTR, which leads to downy mildew resistance, by using tissue culture or by using vegetative propagation. The tissue culture can be selected or derived from any part of the plant, in particular from a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed, or a stem, and can be regenerated into a plant comprising the modified MARCH gene. The regenerated plant also comprises said gene and has downy mildew resistance. Vegetative propagation from a plant part of a plant comprising a modified MARCH gene of the invention will also lead to a new plant comprising said gene, which has downy mildew resistance. Said method preferably relates to the production of a Cucumis sativus plant.

[0064] The present invention relates to a method for identification of a plant comprising a modified MARCH gene of the invention, in particular comprising a MARCH gene comprising a modified 3’UTR, which leads to downy mildew resistance. The identification comprises determining the presence of a modification in SEQ ID No. 1 of the MARCH gene, that leads to a modification in SEQ ID No. 2, which leads to downy mildew resistance. The identification alternatively comprises determining the presence of a modification in SEQ ID No. 3 of the MARCH gene that leads to downy mildew resistance. The identification also comprises determining the presence of a modification in a homologous sequence of SEQ ID No. 1 , SEQ ID No. 3, or SEQ ID No. 2. Alternatively, the identification comprises determining the presence of a modified MARCH gene as disclosed herein. The determination of the presence of a modification, or the presence of a modified gene, is optionally followed by analysing the identified plant for resistance to downy mildew. Said method relates to the identification of a plant of a species belonging to the Cucurbitaceae, and in particular to the identification of a plant of a species belonging to the genus Cucumis. The method preferably relates to the identification of a plant of the species Cucumis sativus.

[0065] Genotypically determining the presence of a modification in the MARCH gene of the invention, comprises identification of any modification in SEQ ID No. 1 and / or SEQ ID No. 3 that leads to downy mildew resistance. A modification in SEQ ID No. 1 will in particular lead to a modification in SEQ ID No. 2 that leads to downy mildew resistance. The presence of a modification is in particular a modification in SEQ ID No. 3 which is the 3’UTR of the MARCH gene. Determining the presence of a modification includes in particular the presence of a C to T modification on position 77 of SEQ ID No. 3, i.e. a C77T SNP, or on the corresponding position of a homologous sequence, which leads to a modified 3’UTR region comprising SEQ ID No. 4, or comprising a homologous sequence of SEQ ID No. 4 that has retained the modification. Determining the presence of a modification can be done through sequence comparison, which is known to the skilled person. Determining a modification is suitably done by using a marker that is designed to identify such modification as its sequence comprises that particular modification. A suitable marker for identification of the modification in the 3’UTR of the present invention comprises a forward primer comprising SEQ ID No. 10 in combination with a reverse primer comprising SEQ ID No. 11.

[0066] Determining the presence of a modification in the MARCH gene that leads to downy mildew resistance phenotypically is performed by comparing the downy mildew resistance of a plant, or a population of plants, comprising the modification in the MARCH gene with the downy mildew resistance of an isogenic plant, or a population of isogenic plants, lacking the modification in the MARCH gene. When the plant, or population of plants, comprising the modification has a higher level of resistance than the plant, or population of plants, lacking the modification, said modification is a modification that leads to downy mildew resistance.

[0067] The present invention further relates to a method for selection of a plant, in particular a Cucumis sativus plant, which has downy mildew resistance, the method comprising identification of a modified MARCH gene of the invention in a plant, and subsequently selecting said plant as a plant which has downy mildew resistance. Optionally, the downy mildew resistance can be confirmed by performing a bio-assay as described in Example 1. The selected plant obtained by such method is also a part of this invention. The plant thus selected can be used as a source to introduce the modified gene into another plant, thereby introducing or improving downy mildew resistance.

[0068] The invention also relates to a method for growing a plant, in particular a Cucumis sativus plant, that has downy mildew resistance or improved downy mildew resistance, the method comprising sowing a seed, in particular a Cucumis sativus seed, that comprises a modified MARCH gene as described herein, and allowing a plant to grow. The invention also relates to propagation material suitable for producing a plant comprising the modified MARCH gene of the invention, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from a microspore, pollen, an ovary, an ovule, an embryo sac, or an egg cell, or is suitable for vegetative reproduction, and is in particular selected from a cutting, a root, a stem cell, or a protoplast, or is suitable for tissue culture of regenerable cells, and is in particular selected from a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed and a stem. The plant produced from the propagation material comprises the modified MARCH gene of the invention that provides downy mildew resistance. A plant of the invention may be used as a source of the propagation material. A tissue culture comprising regenerable cells is also propagation material and also forms a part of this invention.

[0069] The invention further relates to a cell of a plant of the invention. Such a cell may either be in isolated form or a part of the complete plant or parts thereof and still forms a cell of the invention, because such a cell comprises the modified MARCH gene of the invention. Each cell of a plant of the invention carries the modified MARCH gene of the invention. A cell of the invention may also be a regenerable cell that can regenerate into a new plant of the invention.

[0070] The invention further relates to plant tissue of a plant of the invention, which comprises the modified MARCH gene of the invention. The tissue can be undifferentiated tissue or already differentiated tissue. Undifferentiated tissue is for example a stem tip, an anther, a petal, or pollen, and can be used in micropropagation to obtain new plantlets that are grown into new plants of the invention. The tissue can also be grown from a cell of the invention.

[0071] The invention moreover relates to progeny of a plant, a cell, a tissue, or a seed of the invention, which progeny comprises the modified MARCH gene of the invention. Such progeny can in itself be a plant, a cell, a tissue, or a seed. As used herein, progeny comprises the first and all further descendants from a cross with a plant of the invention, wherein a cross comprises a cross with itself or a cross with another plant, and wherein a descendant that is determined to be progeny comprises a modified MARCH gene of the invention. The progeny can in particular be progeny of a plant of the invention deposited under NCIMB number 44324. Descendants can be obtained through selfing and / or further crossing of the deposit. Progeny also encompasses material that is obtained by vegetative propagation or another form of multiplication.

[0072] In one embodiment, the MARCH gene of the invention as used herein is a MARCH gene, comprising a CDS according to SEQ ID No.l or a homologous sequence thereof having at least 70% sequence identity, and comprising a modified 3’UTR of which the wildtype comprises SEQ ID No.3. In one embodiment, the modified 3’UTR is a sequence according to SEQ ID No. 4.

[0073] In any of the embodiments or aspects of the invention that are described herein for a modified MARCH gene, the modified MARCH gene can be combined with another downy mildew resistance conferring gene, in particular with a modified Malectin-like-RLK gene and / or a modified CsAAP2 gene, to obtain improved and / or more durable downy mildew resistance.

[0074] The present invention relates to the use of a modified MARCH gene as described herein for obtaining resistance to downy mildew in a plant, preferably in a Cucurbitaceae plant, most preferably in a Cucumis sativus plant. The invention also relates to the use of a modified MARCH gene as described herein for enhancing, or improving, resistance to downy mildew in a plant that comprises one or more other downy mildew resistance conferring genes, preferably in a Cucurbitaceae plant, most preferably in a Cucumis sativus plant. Said enhancement or improvement is done through introduction of a modified MARCH gene into a plant that already comprises one or more other downy mildew resistance conferring genes, in particular a Malectin- like-RLK gene and / or a modified CsAAP2 gene, or by modifying the endogenous MARCH gene in such a plant into a gene that confers resistance.

[0075] The invention also relates to a marker for the identification of a modified MARCH gene in a plant. A marker of the invention is in particular a marker comprising, and thereby suitable for identifying, a SNP modification, i.e. a polymorphism, on position 77 of SEQ ID No. 3, or on a homologous sequence thereof. Said marker is preferably designed to identify the presence of the T on position 77 of SEQ ID No. 4. Said marker is suitably a marker comprising forward primer SEQ ID No. 10 and reverse primer SEQ ID No. 11, which combination identifies the presence of a C to T modification on position 77 of SEQ ID No. 3. This combination thereby identifies the presence of SEQ ID No. 4, in particular the presence of the T on position 77.

[0076] The invention also relates to a marker for the identification of a modified Malectin- like-RLK gene in a plant. A marker of the invention is in particular a marker comprising, and thereby suitable for identifying, a SNP modification, i.e. a polymorphism, on position 2063 of SEQ ID No. 5, or on a homologous sequence thereof. Said marker is preferably designed to identify the presence of the G on position 2063 of SEQ ID No. 7. Said marker is suitably a marker comprising forward primer SEQ ID No. 13 and reverse primer SEQ ID No. 14, which combination identifies the presence of a C to G modification on position 2063 of SEQ ID No. 5. This combination thereby identifies the presence of SEQ ID No. 7, in particular the presence of the G on position 2063 of SEQ ID No. 7.

[0077] The invention also relates to the use of a marker for identification of a modified MARCH gene or a modified Malectin-like-RLK gene. The invention further relates to the use of a marker, in particular a marker as described herein, for identification of a modified MARCH gene or a modified Malectin-like-RLK gene that leads to downy mildew resistance in a Cucumis sativus plant and / or for selection of a Cucumis sativus plant comprising a modified MARCH gene and / or a modified Malectin-like-RLK gene that leads to downy mildew resistance. The invention in particular relates to the use of a marker designed to identify the presence of a T on position 77 of SEQ ID No. 4. Said marker suitably comprises primers having SEQ ID Nos. 9 and 11 for identification of a wildtype MARCH gene and primers having SEQ ID Nos. 10 and 11 for identification of a modified MARCH gene, or any other marker designed for identification of the C77T SNP in the 3’UTR of said gene. The invention also relates to the use of a marker designed to identify the presence of a G on position 2063 of SEQ ID No. 7. Said marker suitably comprises primers having SEQ ID Nos. 12 and 14 for identification of a wildtype Malectin-like-RLK gene and primers having SEQ ID Nos. 13 and 14 for identification of a modified Malectin-like-RLK gene, or any other marker designed for identification of the C2063G SNP in said gene. The invention also relates to selection of a plant identified by a marker as described herein and to the plant thus selected.

[0078] “Marker” as used herein is intended to refer both to a physical probe that comprises the modification and is thus suitable for detecting said modification in an hybridization experiment and to the actual modification that can be detected by other means, such as sequencing of the gene.

[0079] The present invention will be further illustrated in the Examples that follow and that are for illustration purposes only. The Examples are not intended to limit the invention in any way. In the Examples and in the application reference is made to the following figures.

[0080] FIGURES

[0081] Figure 1 - Nucleotide sequences: SEQ ID No. 1 (wildtype CDS sequence of the MARCH gene of Cucumis sativus), SEQ ID No. 3 (wildtype 3’UTR sequence of the MARCH gene of Cucumis sativus), SEQ ID No. 4 (modified 3’UTR sequence of the MARCH gene of Cucumis sativus), SEQ ID No. 5 (wildtype genomic sequence of the Malectin-like-RLK gene of Cucumis sativus), SEQ ID No. 7 (modified genomic sequence of the Malectin-like-RLK gene of Cucumis sativus).

[0082] Figure 2 - Amino acid sequences: SEQ ID No. 2 (the wildtype MARCH protein encoded by SEQ ID No. 1), SEQ ID No. 6 (wildtype Malectin-like-RLK protein encoded by SEQ ID No. 5), SEQ ID No. 8 (truncated Malectin-like-RLK protein encoded by SEQ ID No. 7).

[0083] Figure 3 - Marker sequences: SEQ ID Nos. 9-11, for identification of a wildtype and a modified MARCH gene, and SEQ ID Nos. 12-14, for identification of a wildtype and a modified Malectin-like-RLK gene. Identification by these markers can at least be done in C. sativus. SEQ ID No. 9 is the forward primer identifying a wildtype MARCH gene, in particular the wildtype 3’UTR of that gene; SEQ ID No. 10 is the forward primer identifying the modification in the 3’UTR resulting in a modified MARCH gene. SEQ ID No. 11 is the common reverse primer for the MARCH gene. SEQ ID No. 12 is the forward primer identifying a wildtype Malectin-like-RLK gene; SEQ ID No. 13 is the forward primer identifying the modification resulting in a modified Malectin-like-RLK gene. SEQ ID No. 14 is the common reverse primer for the Malectin-like-RLK gene.

[0084] DEPOSIT

[0085] Seed of cucumber Cucumis sativus was deposited with NCIMB Ltd, Wellheads Place, Dyce, Aberdeen AB21 7GB United Kingdom on 3 January, 2024, under deposit accession number NCIMB 44324. Seed of NCIMB 44324 comprises a modified MARCH gene, comprising SEQ ID No. 1 and SEQ ID No. 4, homozygously. Seed of NCIMB 44324 further comprises a modified Malectin-like-RLK gene, comprising SEQ ID No. 7, homozygously.

[0086] EXAMPLES

[0087] EXAMPLE 1

[0088] Bio-assay for fine-mapping of a QTL region on chromosome 4 in Cucumis sativus

[0089] During bio-assays of internal genetic resources and internally developed lines, two sources, SOURCE-TO and SOURCE-M4, were identified that showed a useful level of downy mildew resistance to the breeder. A QTL mapping study was performed for both, which led to the identification of a QTL region on chromosome 4.

[0090] For fine-mapping of the region, a population of segregating plants from a cross between a susceptible line, RZ-SUSC, and SOURCE-TO, with recombinations in the identified region, were placed in a field bio-assay. Since downy mildew is abundantly present in many locations, and artificial inoculation is usually rather complicated, the preference is to test downy mildew in the field. In this way, also the essential field resistance can immediately be observed. The presence of downy mildew can be determined by including known susceptible controls, that have reliable symptom development.

[0091] Seeds of F4 populations derived from the cross were sown early July, 2022, in trays, with around 20 plants per population. After one week the plants were transplanted in a field in Germany on 13 July. Natural downy mildew infection took place, and the first observation started when plants of susceptible control Liszt Fl RZ showed clear symptoms, at around 4 weeks after transplanting.

[0092] Scoring was done according to the symptoms as described in Table 1. Table 1 - phenotypic downy mildew scoring

[0093] The F4 populations were genotyped with a large number of markers based on polymorphisms in the initially identified chromosome 4 QTL region. Phenotyping scores from 3 observations, on 22, 26, and 31 of August were related to the marker scores (Table 2). Based on these observations, it was determined that populations with a recombination between markers KASP1 (22147233 bp) and KASP2 (22743655 bp) showed a clear difference in resistance level. It was therefore concluded that the resistance causing part of the QTL had to be located on the right side of KASP1.

[0094] Table 2 - phenotypic scores of F4 populations with recombination

[0095] EXAMPLE 2

[0096] Identification of a downy mildew resistance gene in Cucumis sativus

[0097] In another fine -mapping round for the QTL on chromosome 4 it was determined that the resistance was present in a region between 22.658.552 bp and 22.804.020 bp on the public genome Cs9930_V3. Although both flanking positions were based on markers that identified variation in genes that were present in the QTL region (Table 3), the determination of the region as such was very surprising, since none of the already known and published genes involved in downy mildew resistance was present in this small region.

[0098] Subsequently an analysis was done to identify all variation that was present in the fine-mapped QTL, and that could be related to an annotated gene. This showed however that the variation was either present in an intron, or was not predicted to lead to an effect on protein level (Table 3). The causal downy mildew resistance gene could therefore still not be determined. Next, closer attention was paid to the C to T SNP in the 3’UTR of a gene annotated as LOC101209559, or Csa_012942, which is described in the NCBI database with the public Cs9930_v3 genome as an ‘uncharacterized protein’ . Additional study on this gene showed the presence of several potentially interesting conserved domains in the encoded protein, such as a RING-variant domain and a RING_CH-C4HC3_MARCH domain, which are present in an overlapping region of the protein. Both are domains involved in membrane-associated processes. Also, an Amino Acid Permease domain was observed to be present, which is involved in amino acid transport. These findings determined that the uncharacterized protein was to be designated as a Membrane associated RCH-ring finger AApermease (MARCH) protein. Again, this conclusion was very surprising, since MARCH genes were thus far not known for their involvement in disease resistance in plants.

[0099] Table 3 - variation in genes in the fine-mapped chromosome 4 region

[0100] EXAMPLE 3

[0101] Identification of the causal gene for downy mildew resistance

[0102] Since the region identified in Example 2 did still not give a good explanation of the cause of the resistance on gene level, further analysis was required. From the observed gene-related variations, the most promising appeared to be the C to T substitution on position 22.743.655 of Cs9930_v3, in the 3’UTR of a MARCH gene, or the C to G substitution on position 22.758.565 in the Malectin-like RLK gene. Even though the latter was present in an intron, it seemed to potentially lead to an alternative earlier splice site, which would result in a frame-shift that led to an early stop codon, and thereby a truncated encoded protein.

[0103] To determine the contribution of either, or both, modified genes, in the field bioassay described in Example 1 also a large number of F5 plants derived from the F4 populations were genotyped. In the F4 and F5 generations, populations were identified that segregated for the SNP in the MARCH gene, and also populations that segregated for the Malectin-like-RLK SNP.

[0104] Three F5 populations were homozygous for the modified MARCH gene, while the modified Malectin-like-RLK gene was absent. Five F5 populations had both the modified MARCH gene and the Malectin-like-RLK gene homozygously present. From each population at least 15 plants were included in another part of the field of the bio-assay described in Example 1 , which were planted in 1, 2, or 3 repetitions, depending on seed availability, with at least 15 plants per repetition (Table 4). As a susceptible control, eight F5 populations from the same cross between RZ-SUSC and SOURCE-TO, which populations lacked both modified genes, were planted. In this way, a good comparison of the contribution of the modified genes in the same genetic background could be observed, since these populations are isogenic.

[0105] Table 4 shows the phenotyping results of the F5 populations. Presence of the susceptible allele is indicated with ‘A’, and presence of the allele from the resistant source is indicated with ‘B’. The markers are the same as the markers presented in Table 2. Although infection was high, a clear and consistent improvement for downy mildew resistance could be observed in the populations having only the modified MARCH gene, as compared to the susceptible isogenic populations. However, the results also indicated that an even better, and more durable, resistance could be obtained when the modified MARCH gene was combined with the modified Malectin-like-RLK gene. This seemed to indicate that actually two genes, present closely to each other in the finemapped QTL region, contributed to the downy mildew resistance of the source.

[0106] Table 4 - phenotyping and genotyping of F5 populations

[0107] EXAMPLE 4 Determination of the contribution of a modified MARCH and / or Malectin-like-RLK gene

[0108] Further to the results described in Example 3, more details about the potential contribution of one or both of the modified genes present in the identified region was desired. Next to the F5 populations assayed in Example 3, which were chosen for their homozygosity of the marker scores, also a number of F5 populations still segregated for these markers. Three of those populations, each represented with two repetitions of 14, 15 or 16 plants per repetition, were phenotyped on a plant-by-plant basis. Observation was done twice, with only 1 day in between. For this per-plant phenotyping a more detailed scale from 1-5 was used, whereby a score 1 is most susceptible, and indicates plants with completely yellow leaves, and score 5 is most resistant, and indicates plants having big spots as well as smaller spots. The average of the two observations is presented in Table 5. Marker score B indicates the presence of the modified gene, while marker score A indicates the presence of the wildtype gene.

[0109] Table 5 - per-plant observations of segregating F5 populations, using a 1-5 scoring table

[0110] The combined phenotyping and genotyping details confirmed the earlier observation, that the presence of a modified MARCH gene gave a level of downy mildew resistance, the presence of a modified Malectin-like-RLK gene also gave a level of downy mildew resistance, and the combination of both modified genes increased the downy mildew resistance even further. The data also nicely showed that the resistance inherits in an intermediate way. The addition of one of the genes heterozygously to the other gene that is homozygously present, which situation is presented as a BH or an HB score in Table 5, increases the level of resistance when compared to BA (only a modified MARCH gene) or AB (only a modified Malectin-like-RLK gene), respectively. However, the BB score, wherein both modified genes are homozygously present, still gives the highest downy mildew resistance level.

[0111] Downy mildew resistance is regarded as a quantitative trait for several crops, and stacking of resistance genes is therefore common practice in these instances. It was therefore very useful to have identified two new genes that could be used, separately or in combination, for conferring downy mildew resistance in cucumber. EXAMPLE 5

[0112] Confirmation of the contribution of the individual genes, and the combination of genes.

[0113] To confirm the findings described in Examples 3 and 4, in 2023 another field bioassay was done with plants of the next generation, having recombinations in the initially fine- mapped QTL region on chromosome 4. 45 F6 populations derived from the described F5 populations were again sown in trays early July, 2023, and transplanted to the field on July 11. For each population, at least 20 plants were transplanted per observation plot, whereby some populations were included in repetitions of 20 plants depending on seed availability. As a susceptible control, the hybrid variety Eizst RZ was included. In addition, also three F6 populations without the modified genes were included, in 2 or 3 repetitions depending on seed availability, which serve as an additional susceptible control.

[0114] Ten plots were planted with F6 populations that had only the modified MARCH gene; nine plots were planted with F6 populations that had only the modified Malectin-like-RLK gene; and eleven plots were planted with F6 populations that had the combination of both modified genes. Phenotyping started at around 5 weeks after transplanting and was done twice a week, according to the scoring presented in Table 1.

[0115] The resulting observations of the phenotyping in combination with the genotyping are presented per population in Table 6. The averages per genotype are presented in Table 7. An A score indicates the absence of the modified gene, a B score indicates the presence of the modified gene. The presence or absence of the genes was determined by the markers having SEQ ID Nos. 9 -11 as primers for the MARCH gene, and SEQ ID Nos. 12-14 as primers for the Malectin-like-RLK gene. The combination of primer pair SEQ ID Nos. 9 and 11 determined the presence of the wildtype MARCH gene and the combination of primer pair SEQ ID Nos. 10 and 11 determined the presence of the modified MARCH gene. The combination of primer pair SEQ ID Nos. 12 and 14 determined the presence of the wildtype Malectin-like-RLK gene and the combination of primer pair SEQ ID Nos. 13 and 14 determined the presence of the modified Malectin-like-RLK gene. The bio-assay on the F6 populations very nicely confirmed the earlier findings.

[0116] Again, the presence of each modified gene individually consistently showed an improved downy mildew resistance level as compared to the susceptible commercial control variety, as well as compared to the susceptible isogenic F6 populations that did not have either of the genes. The presence of either the modified MARCH gene or the modified Malectin-like-RLK gene resulted in a comparable downy mildew resistance level. The presence of the combination of both modified genes showed that initially the difference was not that large, but the development of the disease over time really slowed down, and is therefore giving a very useful more durable resistance. Table 6 - observations F6 populations

[0117] Table 7 - averages per genotype

[0118] EXAMPLE 6

[0119] Stacking of a modified MARCH gene, a modified Malectin-like-RLK gene, and a modified CsAAP2 gene.

[0120] Breeding for downy mildew resistance is generally done by combining various genes that each contribute to a resistance level, and together result in a higher and more durable level of resistance. This type of resistance is more robust in field conditions, and less easily broken. To determine the possibility of combining the new genes that were found in the research for this invention with a known downy mildew resistance gene, stacking with a modified CsAAP2 gene was done. The CsAAP2 gene is one of the causal genes on chromosome 4 for the downy mildew resistance originating from PI197088 (Liu et al., supra), and is the subject of patent application WO2020239495 which is based on the PI197088 source. A marker based on the TE insertion in this gene, which insertion is described in said patent application, was developed to check on and follow the presence of a modified CsAAP2 gene having the disclosed TE insertion in exon 4.

[0121] From the start of the research, in parallel, populations combining all three modified genes were developed, and compared in the above described field-bio-assays with populations having only the CsAAP2 gene and populations having the combination of the MARCH gene and the Malectin-like-RLK gene. Results are presented in Table 8. All F4, F5, and F6 lines are isogenic lines with or without the indicated modified genes. The Susceptible F4, F5, and F6 lines are also isogenic, having none of the modified genes. A score A indicates the absence of a modified gene; a score B indicates the presence of a modified gene.

[0122] Table 8 - downy mildew resistance in lines having stacked genes

[0123] The conclusion from the field observations was that, on its own, the presence of a modified CsAAP2 gene having a TE insertion in exon 4 as described in WO2020239495 resulted in an improvement in the level of downy mildew resistance. The combination of all three modified genes, namely a modified MARCH gene, a modified Malectin-like-RLK gene, and a modified CsAAP2 gene led to the highest, and most durable level of resistance.

[0124] EXAMPLE 7

[0125] Modification of a MARCH gene to obtain resistance to downy mildew

[0126] Modifications are introduced in seed of a plant of interest in which resistance, or improved resistance, to downy mildew is desired. The modification is introduced through mutagenesis, such as an EMS treatment, through radiation means, or through a specific targeted approach, such as a CRISPR-CAS method. When a non-targeted approach such as EMS is used, this is combined with an identification technique such as TILLING. In this way, both for mutagenesis as well as a targeted modification means, a modification in a MARCH gene can be generated and identified. The skilled person is familiar with these means for introducing modifications into the genome of a plant of interest.

[0127] Modified seed is then germinated and plants are grown, which are crossed or selfed to generate M2 seed. Subsequently a plant screen is performed to identify the modifications in a MARCH gene, based on comparison to the wildtype sequence of the one or more MARCH genes of that species. For Cucumis sativus for example, comparison to SEQ ID No. 1 (CDS) and SEQ ID No. 3 (3’UTR) can be done. The skilled person is familiar with TILLING to identify mutations in specific genes (McCallum et. al. (2000) Nature Biotechnology, 18: 455-457), and with techniques for identifying nucleotide changes such as DNA sequencing, amongst others. Plants with a modified MARCH gene are heterozygous, homozygous, or can be made homozygous by selfing, crossing, or the use of doubled haploid techniques which are familiar to the skilled person. When non-targeted mutagenesis is used, plants are preferably backcrossed for a number of generations, at least 2, 3, or 4 generations, to get rid of other random mutations in the genome besides the mutation in the MARCH gene. Plants identified and selected on the basis of a modification in a MARCH gene can then be tested for resistance to downy mildew. Preferably, the resistance is compared to the resistance of the non-mutated parent plant. A plant that is produced, identified and selected in this way is confirmed to have their downy mildew resistance, or improved downy mildew resistance, as a result from one or more modifications in the MARCH gene.

Claims

CLAIMS1. Modified MARCH gene, comprising a coding sequence and a 3’UTR, wherein the wildtype of the coding sequence comprises SEQ ID No. 1 or a homologous sequence having at least 70% sequence identity to SEQ ID No. 1 and the wildtype of the 3’UTR comprises SEQ ID No. 3, or a homologous sequence having at least 70% sequence identity to SEQ ID No. 3, which modified gene leads to resistance to downy mildew.

2. Modified MARCH gene as claimed in claim 1, which comprises a modification that leads to an altered gene expression.

3. Modified MARCH gene as claimed in claim 2, wherein the altered gene expression is a reduced gene expression.

4. Modified MARCH gene as claimed in any of the claims 1 to 3, which comprises a modification in SEQ ID No. 3, or in a homologous sequence having at least 70% sequence identity to SEQ ID No. 3.

5. Modified MARCH gene as claimed in claim 4, which comprises a modification on position 77 of SEQ ID No. 3, or a modification on the corresponding position of a homologous sequence having at least 70% sequence identity.

6. Modified MARCH gene as claimed in claim 5, which comprises SEQ ID No. 4.

7. Polynucleotide comprising a modified MARCH gene according to any of the claims 1 to 6 and a modified Malectin-like-RLK gene, preferably comprising SEQ ID No. 4 and SEQ ID No. 7.

8. Plant comprising a modified MARCH gene according to any of the claims 1 to 6.

9. Plant as claimed in claim 8, which is resistant to downy mildew.

10. Plant as claimed in claim 8 or 9, which is a plant of the family Cucurbitaceae, preferably a plant of the species Cucumis sativus.

11. Cucumis sativus plant as claimed in claim 10, wherein the modified MARCH gene is as comprised in the genome of a Cucumis sativus plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 44324.

12. Cucumis sativus plant as claimed in claim 10, wherein the 3’UTR of the modified MARCH gene is as comprised in the genome of a Cucumis sativus plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 44324.

13. Cucumis sativus plant as claimed in any of the claims 10 to 12, which further comprises a modified Malectin-like-RLK gene.

14. Plant as claimed in claim 13, wherein the modified Malectin-like-RLK gene comprises SEQ ID No. 7.

15. Plant as claimed in claim 14, which comprises the polynucleotide of claim 7.

16. Plant as claimed in any of the claims 13 to 15, which has a higher phenotypic score for downy mildew resistance as compared to an isogenic plant comprising only a modified MARCH gene.

17. Seed, comprising a modified MARCH gene according to any of the claims 1 to 6.

18. Seed that can grow into a plant as claimed in any of claims 8 to 16.

19. Marker for the identification of a modified MARCH gene, wherein the marker detects a modification on position 77 of SEQ ID No. 3, or wherein the marker detects a modification on a corresponding position of a homologous sequence having at least 70% sequence identity to SEQ ID No. 3.

20. Marker as claimed in claim 19 which comprises SEQ ID No. 10, optionally in combination with SEQ ID No. 11.

21. Marker for the identification of a modified Malectin-like-RLK gene, which marker preferably comprises SEQ ID No. 13, optionally in combination with SEQ ID No. 14.

22. Use of a marker as claimed in any of claims 19 to 21 for identification of a modified MARCH gene or a modified Malectin-like-RLK gene that leads to downy mildew resistance in a Cucumis sativus plant and / or for selection of a downy mildew resistant Cucumis sativus plant.

23. Method for producing a downy mildew resistant Cucumis sativus plant comprising introducing a modification in a MARCH gene, wherein the MARCH gene comprising the modification is a gene according to any of the claims 1 to 6.

24. Method for selecting a downy mildew resistant Cucumis sativus plant, comprising identifying the presence of a modification in a MARCH gene, optionally testing the plant for downy mildew resistance, and selecting a plant that comprises said modification as a downy mildew resistant plant.

25. Method as claimed in claim 24, wherein the modification to be identified comprises a C to T mutation on position 77 of SEQ ID No. 3, or comprises the presence of a T on position 77 of SEQ ID No. 4.

26. Method as claimed in claim 24 or 25, wherein the identification is performed by using a marker as defined in claim 19 or 20.

27. Method for the production of a plant which is resistant to downy mildew, said method comprising: a) crossing a first parent plant as claimed in any of the claims 8 to 16, with a second parent plant; b) optionally performing one or more rounds of selfing and / or crossing of the plant resulting from the cross in step a) to obtain a further generation population;c) selecting from the plant resulting from the cross in step a), or from the further generation population of step b), a plant that comprises a modified MARCH gene, wherein the selected plant is resistant to downy mildew.

28. Method for the production of a Cucumis sativus plant which is resistant to downy mildew, said method comprising: a) crossing a first parent plant as claimed in any of the claims 10 to 16, with a second parent plant; b) optionally performing one or more rounds of selfing and / or crossing of the plant resulting from the cross in step a) to obtain a further generation population; c) selecting from the plants resulting from the cross in step a), or from the further generation population of step b), a plant that comprises a modified MARCH gene, wherein the selected plant is resistant to downy mildew.

29. Method as claimed in claim 27 or 28, wherein the second parent plant also comprises a modified MARCH gene.

30. Method as claimed in any of the claims 27 to 29, wherein selection of a plant comprising a modification in a MARCH gene is performed by using a marker as claimed in claim 19 or 20.

31. Method as claimed in any of the claims 27 to 29, wherein a plant which is resistant to downy mildew is phenotypically selected, in particular by using a bio-assay for downy mildew resistance.

32. Method as claimed in any of the claims 27 to 31 , wherein the plant as claimed in any of the claims 10 to 16 is a plant grown from seed deposited under NCIMB deposit number NCIMB 44324, or a progeny plant thereof that has retained the modified MARCH gene.

33. Method for the production of hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and / or the second parent plant is a plant comprising a modified MARCH gene according to any of the claims 1 to 6, and wherein the presence of said modified MARCH gene leads to downy mildew resistance in a plant that is grown from the hybrid seed.

34. Method as claimed in claim 33, wherein the hybrid seed is seed of the species Cucumis sativus.

35. Method for growing a plant, in particular a Cucumis sativus plant, that has downy mildew resistance, the method comprising sowing a seed, in particular a Cucumis sativus seed, comprising a modified MARCH gene according to any of claims 1 to 6, and allowing a plant to grow.

36. Use of a modified MARCH gene according to any of claims 1 to 6 for obtaining resistance to downy mildew in a plant, preferably in a Cucurbitaceae plant, most preferably in a Cucumis sativus plant.

37. Use of a modified MARCH gene according to any of claims 1 to 6 for improving resistance to downy mildew in a plant that comprises one or more other downy mildew resistance conferring genes, preferably in a Cucurbitaceae plant, most preferably in a Cucumis sativus plant.

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