Rijk Zwaan Zaadteelt En Zaadhandel B.v.

Raoul Jacobus Johannes Maria Frijters

Vincent Laurens Adrianus Kock

Johannes Geert Jan Feitsma

Jasper De Joode

Erik Den Boer

Cornelis Maria Petrus Van Dun

Cornelis Haaring

Lena Johanna Huijbregts-Doorduin

Sara Movahedi

Japser De Joode

Eric Cornelis Josephus Bal

Paul De Graag

The present invention relates to a modified gene that provides resistance to Cucurbit Yellow Stunting Disorder virus (CYSDV) when present in a plant of the Cucurbitaceae family. The invention further relates to progeny, seed, and fruit of the plant that is resistant against CYSDV. The invention also relates to propagation material suitable for producing the plant that is resistant to CYSDV. Additionally, the invention relates to methods for producing, identifying, and selecting a plant of the Cucurbitaceae family having resistance against CYSDV.

CYSDV resistance in members of the cucurbitaceae family

The present invention relates to an allele designated alpha-CMV which confers resistance to CMV when present in a spinach plant, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif 'MAEIGYSVC' (SEQ ID NO: 22) at its N-terminus; and b) the motif “KWMCLR” (SEQ ID NO: 23); and c) an LRR domain that has in order of increased preference at least 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 10. The allele has a genomic nucleotide sequence which in order of increased preference has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 1. The invention further relates to plants comprising the allele and to various methods involving the allele.

CMV resistance allele

The present invention relates to an allele designated alpha-WOLF 26 which confers resistance to at least onef. sp.race, wherein the protein encoded by said allele is a CC-NBS-LRR protein that may comprise in its amino acid sequence: a) the motif 'MAEIGYSVC' SEQ ID NO: 1 at its N-terminus; and b) the motif 'KWMCLR' SEQ ID NO: 2; and wherein the LRR domain of the protein has in order of increased preference at least 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% sequence identity to SEQ ID NO: 10. The allele when present in a spinach plant confers complete resistance to at leastf. sp.race Pfs:7, Pfs:10, Pfs:11, Pfs:12, Pfs:13, Pfs:14, Pfs:15, Pfs:16, Pfs:17, and does not confer resistance to downy mildew race Pfs:8 and Pfs:9.

resistance in

The present invention relates to an allele designated alpha-WOLF 25 which confers resistance to at least onef. sp.race, wherein the protein encoded by said allele is a CC-NB S-LRR protein that comprises in its amino acid sequence: a) the motif 'MAEIGYSVC' at its N-terminus; and b) the motif 'KWMCLR'; and wherein the LRR domain of the protein has in order of increased preference at least 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 5. The allele when present in a spinach plant confers complete resistance to at leastf sp.race Pfs:8, Pfs15 and Pfs:16, and does not confer resistance to Pfs:3.

The present invention relates to an allele designated alpha-WOLF 27 which confers resistance to at least onef. sp.race, wherein the protein encoded by said allele is a CC-NBS-LRR protein that may comprise in its amino acid sequence: a) the motif 'MAEIGYSVC' SEQ ID NO: 1 at its N-terminus; and b) the motif 'KWMCLR' SEQ ID NO: 2; and wherein the LRR domain of the protein has in order of increased preference at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% sequence similarity to SEQ ID NO: 10. The allele when present in a spinach plant confers complete resistance to at leastf. sp.race Pfs:7, Pfs:8, Pfs:9, Pfs:11, Pfs:12, Pfs:13, Pfs:14, Pfs:15, Pfs:17.

The present invention relates to a genetic determinant which may comprise at least two copies of a combination of two closely linked RDR1 genes, which two closely linked RDR1 genes are inversely oriented, and which genetic determinant leads to virus resistance when present in a plant. In one embodiment, of the RDR1 genes in the combination is represented by SEQ ID NO: 1 or has at least 70% sequence identity, and one of the RDR1 genes in the combination is represented by SEQ ID NO: 3 or has at least 70% sequence identity; or one of the RDR1 genes in the combination encodes a protein represented by SEQ ID NO: 2 or a protein that has at least 70% sequence identity, and one of the RDR1 genes encodes a protein represented by SEQ ID NO: 4 or a protein that has at least 70% sequence identity.

Copy number variant leading to virus resistance

The present invention relates to a method for modifying the resistance profile of a spinach plant tof sp., comprising introducing a WOLF allele or a resistance-conferring part thereof into the genome of said spinach plant, or modifying an endogenous WOLF allele in the genome of said spinach plant. A WOLF allele encodes a CC-NBS-LRR protein that comprises in its amino acid sequence: the motif 'MAEIGYSVC' at its N-terminus, and the motif 'KWMCLR' for alpha-type WOLF proteins or &#x201c;HVGCVVDR&#x201d; for beta-type WOLF proteins. The invention relates to the WOLF alleles referred to in Table 3. The invention further provides a method for selecting a spinach plant comprising a novel WOLF gene that confers resistance tof sp.in a spinach plant and a method for identifying a WOLF allele that confers resistance to one or more pathogenic races off. sp.in a spinach plant and to primers for use in these methods.

Method for modifying the resistance profile ofto downy mildew

The present invention relates to a method for modifying the resistance profile of a spinach plant tof. sp., comprising introducing a WOLF allele or a resistance-conferring part thereof into the genome of said spinach plant, or modifying an endogenous WOLF allele in the genome of said spinach plant. The invention further provides a method for selecting a spinach plant comprising a novel WOLF gene that confers resistance tof. sp.in a spinach plant and a method for identifying a WOLF allele that confers resistance to one or more pathogenic races off. sp.in a spinach plant and to primers for use in these methods.

The present invention relates to an allele designated alpha-WOLF 8 which confers resistance to at least onef. Sp.race, wherein the protein encoded by said allele is a CC-NB S-LRR protein that comprises in its amino acid sequence: a) the motif 'MAEIGYSVC' (SEQ ID NO: 15) at its N-terminus; and b) the motif 'KWMCLR' (SEQ ID NO: 16); and wherein the LRR domain of the protein has in order of increased preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 12. When the allele is homozygously present in a spinach plant confers complete resistance tof. Sp.races pfs:1, pfs:2, pfs:6, pfs:8 and pfs:15, and isolate US1508, and confers intermediate resistance to pfs:5, pfs:10 and pfs:16, and does not confer resistance to pfs:3, pfs:4, pfs:7, pfs:9, pfs:11, pfs:12, pfs:13 and pfs:14.

The present invention relates to an allele designated alpha-WOLF 15 which confers resistance to at least onef. Sp.race, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif 'MAEIGYSVC' (SEQ ID NO: 13) at its N-terminus; and b) the motif 'KWMCLR' (SEQ ID NO: 14); and wherein the LRR domain of the protein has in order of increased preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 10. The allele when homozygously present in a spinach plant confers complete resistance tof. Sp.races pfs:1, pfs:2, pfs:3, pfs:4 and pfs:5, pfs:6, pfs:8, pfs:9, pfs:11, pfs:12, pfs:13, pfs:14, pfs:15, pfs:16 and isolates UA1014 and US1508, and confers intermediate resistance to pfs:10, and does not confer resistance to pfs:7.

The present invention relates to an allele designated alpha-CMV which confers resistance to CMV when present in a spinach plant, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif 'MAEIGYSVC' (SEQ ID NO: 22) at its N-terminus; and b) the motif 'KWMCLR' (SEQ ID NO: 23); and c) an LRR domain that has in order of increased preference at least 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 10. The allele has a genomic nucleotide sequence which in order of increased preference has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 1. The invention further relates to plants comprising the allele and to various methods involving the allele.

The present invention relates to a genetic determinant which may comprise at least two copies of a combination of two closely linked RDR1 genes, which two closely linked RDR1 genes are inversely oriented, and which genetic determinant leads to virus resistance when present in a plant. In one embodiment, of the RDR1 genes in the combination is represented by SEQ ID No. 1 or has at least 70% sequence identity, and one of the RDR1 genes in the combination is represented by SEQ ID No. 3 or has at least 70% sequence identity; or one of the RDR1 genes in the combination encodes a protein represented by SEQ ID No. 2 or a protein that has at least 70% sequence identity, and one of the RDR1 genes encodes a protein represented by SEQ ID No. 4 or a protein that has at least 70% sequence identity.

The present invention relates to an allele designated alpha-WOLF 8 which confers resistance to at least onef. sp.race, wherein the protein encoded by said allele is a CC-NB S-LRR protein that comprises in its amino acid sequence: a) the motif 'MAEIGYSVC' (SEQ ID NO: 15) at its N-terminus; and b) the motif 'KWMCLR' (SEQ ID NO: 16); and wherein the LRR domain of the protein has in order of increased preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID NO: 12. When the allele is homozygously present in a spinach plant confers complete resistance tof. sp.races Pfs:1, Pfs:2, Pfs:6, Pfs:8 and Pfs:15, and confers intermediate resistance to Pfs:5, Pfs:10 and Pfs:16, and does not confer resistance to Pfs:3, Pfs:4, Pfs:7, Pfs:9, Pfs:11, Pfs:12, Pfs:13 and Pfs:14.

resistance in spinacia oleracea

The present invention relates to a method for modifying the resistance profile of a spinach plant tof. sp., comprising introducing a WOLF allele or a resistance-conferring part thereof into the genome of said spinach plant, or modifying an endogenous WOLF allele in the genome of said spinach plant. A WOLF allele encodes a CC-NBS-LRR protein that comprises in its amino acid sequence: the motif 'MAEIGYSVC' at its N-terminus, and the motif 'KWMCLR' for alpha-type WOLF proteins or &#x201c;HVGCVVDR&#x201d; for beta-type WOLF proteins. The invention relates to the WOLF alleles referred to in Table 3. The invention further provides a method for selecting a spinach plant comprising a novel WOLF gene that confers resistance tof. sp.in a spinach plant and a method for identifying a WOLF allele that confers resistance to one or more pathogenic races off. sp.in a spinach plant and to primers for use in these methods.

The present invention relates to modified RDR1 gene capable of conferring virus resistance to a plant and/or increasing virus resistance in a plant, which modification results in enhanced expression of the RDR1 gene, and wherein the modification is selected from a modification that increases the mRNA level of the RDR1 gene; a modification that increases the level of the RDR1 protein; and/or a modification that increases the activity of the RDR1 protein, as compared to a non-modified wild-type RDR1 gene. The modification may comprise a modification upstream of the coding sequence of the RDR1 gene, such as a modification of a regulatory element, preferably of a cis-acting regulatory element. The modified regulatory element is for example selected from a transcription factor binding site for a transcriptional repressor, the modification of which leads to reduction or absence of transcriptional repression; and/or a transcription factor binding site for a transcriptional activator, the modification of which leads to induction or enhancement of transcription; and/or a microRNA binding site, the modification of which leads to reduction or absence of gene repression; and/or a small RNA sequence, the modification of which leads to reduction or absence of gene repression.

Growing community of inventors

De Lier, Netherlands

Raoul Jacobus Johannes Maria Frijters