# US 2023/0056618 A1 · Cmv resistance conferring genes

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- Number: US 2023/0056618 A1
- Document ID: US20230056618A1
- Status: Published application
- Kind: A1
- Application number: 17/875,549
- Filed: 2022-07-28
- Published: 2023-02-23
- Earliest claimed: 2020-02-26
- Applicants: Rijk Zwaan Zaadteelt En Zaadhandel B.V.
- Inventors: Cornelis VAN DER MAAS; Adrianus Cornelis KOEKEN
- Web page: https://patentgrep.com/patent/US20230056618A1

## CPC

- A01H 6/346: {Cucumis sativus[cucumber]}
- C12N 15/8283: {for virus resistance}
- A01H 5/08: Fruits
- C07K 14/415: from plants
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/13: Plant traits
- C12Q 2600/156: Polymorphic or mutational markers
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to a modified ABCB9 gene encoding a protein conferring resistance to CMV in a plant of the Cucurbitaceae, such as a cucumber plant, in which the protein is expressed, where the gene has a) a nucleotide sequence which encodes a protein with SEQ ID NO: 4; b) a nucleotide sequence with SEQ ID NO: 3; c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein with SEQ ID NO: 4; d) a nucleotide sequence that encodes a protein with an amino acid sequence, which is at least 85% identical to SEQ ID NO: 4; e) a nucleotide sequence which is at least 85% identical to SEQ ID NO: 3; or f) a nucleotide sequence according to c) or d) wherein the protein has a methionine (M) on position 428 of SEQ ID NO: 4, or on a position corresponding thereto.

## Claims

### Claim 1

A modified ABCB9 gene encoding a protein conferring resistance to CMV in a plant of the Cucurbitaceae, in which the protein is expressed, characterized in that the gene comprises a nucleotide sequence of
a) a nucleotide sequence which encodes a protein comprising SEQ ID NO: 4;
b) a nucleotide sequence comprising SEQ ID NO: 3;
c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein comprising SEQ ID NO: 4;
d) a nucleotide sequence that encodes a protein comprising an amino acid sequence, which is at least 85% identical to SEQ ID NO: 4;
e) a nucleotide sequence which is at least 85% identical to SEQ ID NO: 3; or
f) a nucleotide sequence according to c) or d) wherein the protein comprises a methionine (M) on position 428 of SEQ ID NO: 4, or on a position corresponding thereto.

### Claim 2 (depends on 1)

A protein conferring resistance to CMV in a plant of the Cucurbitaceae, in which the protein is expressed and which protein is encoded by the gene of claim 1.

### Claim 3

A modified EF1-α gene encoding a protein conferring resistance to CMV in a cucumber plant, in which the protein is expressed, characterized in that the gene comprises a nucleotide sequence of
a) a nucleotide sequence which encodes a protein comprising SEQ ID NO: 8;
b) a nucleotide sequence comprising SEQ ID NO: 7;
c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein comprising SEQ ID NO: 8, d) a nucleotide sequence that encodes a protein comprising an amino acid sequence, which is at least 95% identical to SEQ ID NO: 8;
e) a nucleotide sequence which is at least 95% identical to SEQ ID NO: 7; or
f) a nucleotide sequence according to c) or d) wherein the protein comprises a Glycine (G) on position 372 of SEQ ID NO: 8, or on a position corresponding thereto.

### Claim 4 (depends on 3)

A protein conferring resistance to CMV in a plant of the Cucurbitaceae, in which the protein is expressed and which protein is encoded by the gene of claim 3.

### Claim 5 (depends on 1)

A plant of the Cucurbitaceae comprising the modified ABCB9 gene of claim 1.

### Claim 6 (depends on 5)

The plant of claim 5 comprising the modified ABCB9 gene homozygously, wherein the plant is resistant to CMV.

### Claim 7 (depends on 5)

The plant of claim 5, further comprising a modified EF1-α gene encoding a protein conferring resistance to CMV in a cucumber plant, in which the protein is expressed, characterized in that the gene comprises a nucleotide sequence of a) a nucleotide sequence which encodes a protein comprising SEQ ID NO: 8; b) a nucleotide sequence comprising SEQ ID NO: 7; c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein comprising SEQ ID NO: 8, d) a nucleotide sequence that encodes a protein comprising an amino acid sequence, which is at least 95% identical to SEQ ID NO: 8; e) a nucleotide sequence which is at least 95% identical to SEQ ID NO: 7; or f) a nucleotide sequence according to c) or d) wherein the protein comprises a Glycine (G) on position 372 of SEQ ID NO:
8, or on a position corresponding thereto.

### Claim 8 (depends on 5)

The plant of claim 5, comprising the modified EF1-α gene, homozygously.

### Claim 9 (depends on 5)

A part of the plant of claim 5, which plant part comprises the modified ABCB9 gene and optionally a modified EF1-α gene encoding a protein conferring resistance to CMV in a cucumber plant, in which the protein is expressed, characterized in that the gene comprises a nucleotide sequence selected from a) a nucleotide sequence which encodes a protein comprising SEQ ID NO: 8; b) a nucleotide sequence comprising SEQ ID NO: 7; c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein comprising SEQ ID NO: 8, d) a nucleotide sequence that encodes a protein comprising an amino acid sequence, which is at least 95% identical to SEQ ID NO: 8; e) a nucleotide sequence which is at least 95% identical to SEQ ID NO: 7; or f) a nucleotide sequence according to c) or d) wherein the protein comprises a Glycine (G) on position 372 of SEQ ID NO: 8, or on a position corresponding thereto.

### Claim 10 (depends on 1)

A seed comprising the modified ABCB9 gene of claim 1, and optionally a modified EF1-α gene encoding a protein conferring resistance to CMV in a cucumber plant, in which the protein is expressed, characterized in that the gene comprises a nucleotide sequence of a) a nucleotide sequence which encodes a protein comprising SEQ ID NO: 8; b) a nucleotide sequence comprising SEQ ID NO: 7; c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein comprising SEQ ID NO: 8, d) a nucleotide sequence that encodes a protein comprising an amino acid sequence, which is at least 95% identical to SEQ ID NO: 8; e) a nucleotide sequence which is at least 95% identical to SEQ ID NO: 7; or f) a nucleotide sequence according to c) or d) wherein the protein comprises a Glycine (G) on position 372 of SEQ ID NO: 8, or on a position corresponding thereto.

### Claim 11 (depends on 5)

A tissue culture of the plant of claim 5, which tissue culture comprises the modified ABCB9 gene, and optionally a modified EF1-α gene encoding a protein conferring resistance to CMV in a cucumber plant, in which the protein is expressed, characterized in that the gene comprises a nucleotide sequence of a) a nucleotide sequence which encodes a protein comprising SEQ ID NO: 8; b) a nucleotide sequence comprising SEQ ID NO: 7; c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein comprising SEQ ID NO: 8, d) a nucleotide sequence that encodes a protein comprising an amino acid sequence, which is at least 95% identical to SEQ ID NO: 8; e) a nucleotide sequence which is at least 95% identical to SEQ ID NO: 7; or f) a nucleotide sequence according to c) or d) wherein the protein comprises a Glycine (G) on position 372 of SEQ ID NO: 8, or on a position corresponding thereto.

### Claim 12

A marker for identifying a cucumber plant comprising a modified ABCB9 gene, wherein the marker detects a modified nucleotide on position 1282 as compared to the wild type ABCB9 gene nucleotide sequence of SEQ ID NO: 1.

### Claim 13 (depends on 12)

The marker of claim 12, wherein the marker detects an Adenine (A) on position 1282 of SEQ ID NO: 3.

### Claim 14 (depends on 13)

The marker of claim 13, comprising SEQ ID NO: 11.

### Claim 15

A marker for identifying a cucumber plant comprising a modified EF1-α gene, wherein the marker detects a modified nucleotide on position 1115 as compared to the wild type EF1-α gene nucleotide sequence of SEQ ID NO: 5.

### Claim 16 (depends on 15)

The marker of claim 15, wherein the marker detects a Guanine (G) on position 1115 of SEQ ID NO: 7.

### Claim 17 (depends on 16)

The marker of claim 16, comprising SEQ ID NO: 12.

### Claim 18

A method of identifying a cucumber plant that comprises a modified ABCB9 gene and/or a modified EF1-α gene, and in particular for identifying a plant that is resistant to CMV, comprising detecting the plant with a marker that detects a modified nucleotide on position 1282 as compared to the wild type ABCB9 gene nucleotide sequence of SEQ ID NO: 1 or a marker that detects a modified nucleotide on position 1115 as compared to the wild type EF1-α gene nucleotide sequence of SEQ ID NO: 5.

### Claim 19 (depends on 1)

A method for producing a plant that is CMV resistant by introducing the modified ABCB9 gene of claim 1 and optionally introducing a modified EF1-α gene encoding a protein conferring resistance to CMV in a cucumber plant, in which the protein is expressed, characterized in that the gene comprises a nucleotide sequence of a) a nucleotide sequence which encodes a protein comprising SEQ ID NO: 8; b) a nucleotide sequence comprising SEQ ID NO: 7; c) a nucleotide sequence encoding a protein derived by substitution, deletion and/or addition of one or more amino acids of the protein comprising SEQ ID NO: 8, d) a nucleotide sequence that encodes a protein comprising an amino acid sequence, which is at least 95% identical to SEQ ID NO: 8; e) a nucleotide sequence which is at least 95% identical to SEQ ID NO: 7; or f) a nucleotide sequence according to c) or d) wherein the protein comprises a Glycine (G) on position 372 of SEQ ID NO: 8, or on a position corresponding thereto into the genome of a plant.

### Claim 20 (depends on 1, 3)

A method for selecting a CMV resistant plant, comprising
a) identifying the presence of the modified ABCB9 gene of claim 1 and/or the presence of the modified EF-1 α gene of claim 3,
b) selecting a plant that comprises at least one of said modified genes;
c) optionally testing the selected plant for CMV resistance, and
d) selecting the plant if it shows CMV resistance as a CMV resistant plant.

### Claim 21 (depends on 20)

The method of claim 20, wherein the identification is performed with a marker for identifying a cucumber plant comprising a modified ABCB9 gene, wherein the marker detects a modified nucleotide on position 1282 as compared to the wild type ABCB9 gene nucleotide sequence of SEQ ID NO: 1 or the marker detects a modified nucleotide on position 1115 as compared to the wild type EF1-α gene nucleotide sequence of SEQ ID NO: 5.

### Claim 22 (depends on 1)

A method for producing a plant that comprises the modified ABCB9 gene of claim 1, said method comprising:
a) crossing a first parent plant comprising the modified ABCB9 gene, of claim 1, with a second parent plant, to obtain an F1 population,
b) optionally performing one or more rounds of selfing and/or crossing with a plant from said F1 population to obtain a further generation population,
c) selecting a plant that comprises the modified ABCB9 gene.

### Claim 23 (depends on 3)

A method for producing a plant that comprises the modified EF1-α gene of claim 3, said method comprising:
a) crossing a first parent plant comprising the modified EF1-α gene, with a second parent plant, to obtain an F1 population,
b) optionally performing one or more rounds of selfing and/or crossing with a plant from said F1 population to obtain a further generation population,
c) selecting a plant that comprises the modified EF1-α gene.

### Claim 24 (depends on 22)

The method of claim 22, further comprising the steps of:
d) testing the selected plant for showing CMV resistance;
e) selecting the plant if it is CMV resistant.

### Claim 25 (depends on 23)

The method of claim 23, further comprising the steps of:
d) testing the selected plant for showing CMV resistance;
e) selecting the plant if it is CMV resistant.

### Claim 26 (depends on 1, 22)

The method of claim 22, wherein the second parent plant also comprises a modified ABCB9 gene of claim 1.

### Claim 27 (depends on 3, 23)

The method of claim 23, wherein the second parent plant also comprises the modified EF1-α gene of claim 3.

### Claim 28 (depends on 3, 22)

The method of claim 22, wherein the first and/or the second parent plant also comprises the modified EF1-α gene of claim 3.

### Claim 29 (depends on 1)

A method for selecting a plant that comprises the modified ABCB9 gene of claim 1 said method comprising;
a) assaying nucleic acids of a plant for the presence of the modified ABCB9 gene of claim 1;
b) identifying a plant that comprises the modified ABCB9 gene, and selecting said plant.

### Claim 30 (depends on 3)

A method for selecting a plant that comprises the modified EF1-α gene, said method comprising;
a) assaying nucleic acids of a plant for the presence of the modified EF1-α gene of claim 3,
b) identifying a plant that comprises the modified EF1-α gene, and selecting said plant.