# US 11,895,961 B2 · CGMMV resistant Citrullus plants

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,895,961 B2
- Document ID: US11895961B2
- Status: Granted
- Kind: B2
- Application number: 17/473,604
- Filed: 2021-09-13
- Published: 2022-02-17
- Granted: 2024-02-13
- Earliest claimed: 2017-08-17
- Assignees: Nunhems B.V.
- Applicants: Nunhems B.V.
- Inventors: Alberto Sirizzotti; Daniel Bellon Dona; Richard Bernard Berentsen; Daniel Jimenez Fernandez; Courtney Hu; Daniel Puglisi
- Web page: https://patentgrep.com/patent/US11895961B2

## CPC

- A01H 6/342: {Citrullus lanatus [watermelon]}
- A01H 1/045: {using molecular markers}
- A01H 1/126: {for virus resistance}
- A01H 5/08: Fruits
- A01H 5/10: Seeds
- 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
- 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 cultivated watermelon plants comprising CGMMV resistance due to introgression of QTLs from a CGMMV resistant donor of the species Citrullus colocynthis.

## Claims

### Claim 1

A cultivated watermelon plant or plant cell comprising an introgression fragment on chromosome 9 and/or on chromosome 7 of a CGMMV resistant donor plant of the species Citrullus colocynthis, wherein the introgression fragment on chromosome 9 comprises a CGMMV resistance QTL from a C. colocynthis donor of which a representative sample has been deposited under accession number NCIMB 42624 and wherein the QTL is in-between marker SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome and marker InDel9.1 at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, and wherein the introgression fragment on chromosome 7 comprises a CGMMV resistance QTL from a C. colocynthis donor of which a representative sample has been deposited under accession number NCIMB 42624 and wherein the QTL is in-between marker InDel7.1 at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome and marker SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome.

### Claim 2 (depends on 1)

A cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 9 comprises a Cytosine for marker SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome and/or comprises a Guanine for marker InDel9.1 instead of a Guanine-Cytosine at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to a deletion of the Cytosine at nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, and wherein the introgression fragment on chromosome 7 comprises an Adenine for marker InDel7.1 instead of an Adenine-Thymine at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome and/or comprises a Adenine for marker SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome.

### Claim 3 (depends on 1)

The cultivated watermelon plant or plant cell according to claim 1, wherein the QTL on chromosome 9 is in-between SNP22 at nucleotide 51 of SEQ ID NO: 22, corresponding to nucleotide 35,343,850 of chromosome 9, and SNP23 at nucleotide 51 of SEQ ID NO: 23, corresponding to nucleotide 36,923,004 of chromosome 9, and wherein the QTL on chromosome 7 is in-between SNP13 at nucleotide 51 of SEQ ID NO: 13, corresponding to nucleotide 4,784,519 of chromosome 7, and SNP16 at nucleotide 51 of SEQ ID NO: 16, corresponding to nucleotide 11,166,694 of chromosome 7.

### Claim 4 (depends on 1)

A cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 7 comprises an Adenine at nucleotide 51 of SEQ ID NO: 14 or an Adenine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 14, and/or a Guanine at nucleotide 51 of SEQ ID NO: 15 or a Guanine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 15.

### Claim 5 (depends on 1)

The cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 7 is in homozygous or in heterozygous form and/or wherein the introgression fragment on chromosome 9 is in homozygous form or in heterozygous form.

### Claim 6 (depends on 1)

The cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment is from a Citrullus colocynthis accession, a representative sample of seeds of the accession having been deposited under accession number NCIMB 42624.

### Claim 7 (depends on 1)

The cultivated watermelon plant or plant cell according to claim 1, wherein the plant comprises a CGMMV resistance of scale 3 (mild symptoms) or scale 4 (no symptoms).

### Claim 8 (depends on 1)

The cultivated watermelon plant or plant cell according to claim 1, wherein the plant or plant cell is an inbred plant or plant cell, or an F1 hybrid plant or plant cell, or a diploid or tetraploid or triploid plant or plant cell.

### Claim 9 (depends on 1)

A seed which grows into a plant according to claim 1.

### Claim 10 (depends on 1)

A cultivated watermelon fruit or fruit part comprising plant cells according to claim 1.

### Claim 11 (depends on 1)

A cultivated watermelon plant propagation material comprising a cultivated watermelon plant cell according to claim 1.

### Claim 12 (depends on 1)

A method for producing triploid hybrid cultivated watermelon seeds comprising the following steps:
a) providing a first CGMMV resistant inbred diploid watermelon plant comprising two chromosomes 7, each having an introgression fragment from chromosome 7 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor;
b) providing a second CGMMV resistant inbred tetraploid watermelon plant comprising four chromosomes 7 each having an introgression fragment from chromosome 7 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor;
c) allowing pollination of the tetraploid watermelon plant provided in step b) with pollen of the diploid watermelon plant provided in step a); and
d) collecting seeds from the fruits produced in step c).

### Claim 13 (depends on 1)

A method for producing triploid hybrid cultivated watermelon seeds comprising the following steps:
a) providing a first CGMMV resistant inbred diploid watermelon plant comprising two chromosomes 9, each having an introgression fragment from chromosome 9 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor;
b) providing a second CGMMV resistant inbred tetraploid watermelon plant comprising four chromosomes 9 each having an introgression fragment from chromosome 9 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor;
c) allowing pollination of the tetraploid watermelon plant provided in step b) with pollen of the diploid watermelon plant provided in step a); and
d) collecting seeds from the fruits produced in step c).

### Claim 14

A method of screening plants or plant parts or seeds, or DNA derived therefrom, for the presence of a fragment on chromosome 7 comprising a CGMMV resistance QTL and/or on chromosome 9 comprising a CGMMV resistance QTL conferring CGMMV resistance comprises the steps of:
a) screening the genomic DNA for the SNP genotype of one or more or all of SNP13 at nucleotide 51 of SEQ ID NO: 13, InDel7.1 at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome, SNP14 at nucleotide 51 of SEQ ID NO: 14, SNP15 at nucleotide 51 of SEQ ID NO: 15, SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome, SNP16 at nucleotide 51 of SEQ ID NO: 16, SNP17 at nucleotide 51 of SEQ ID NO: 17, SNP18 at nucleotide 51 of SEQ ID NO: 18, linked to the QTL on chromosome 7, and/or for one or more of SNP19 at nucleotide 51 of SEQ ID NO: 19, SNP20 at nucleotide 51 of SEQ ID NO: 20, SNP21 at nucleotide 51 of SEQ ID NO: 21, SNP22 at nucleotide 51 of SEQ ID NO: 22, SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome, InDel9.1 at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, SNP23 at nucleotide 51 of SEQ ID NO: 23 and SNP24 at nucleotide 51 of SEQ ID NO: 25, linked to the QTL on chromosome 9; and
b) optionally selecting a plant or plant part which comprises on chromosome 9 a Cytosine for marker SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome and/or a Guanine for marker InDel9.1 instead of a Guanine-Cytosine at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to a deletion of the Cytosine at nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, or selecting a plant or plant part which comprises on chromosome 7 an Adenine for marker InDel7.1 instead of an Adenine-Thymine at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome and/or an Adenine at nucleotide 51 of SEQ ID NO: 14 or an Adenine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 14, and/or a Guanine at nucleotide 51 of SEQ ID NO: 15 or a Guanine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 15, and/or an Adenine for marker SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome.

### Claim 15 (depends on 2)

A cultivated watermelon plant or plant cell according to claim 2, wherein the introgression fragment on chromosome 7 comprises an Adenine at nucleotide 51 of SEQ ID NO: 14 or an Adenine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 14, and/or a Guanine at nucleotide 51 of SEQ ID NO: 15 or a Guanine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 15.