# US 10,767,190 B2 · Brassicaceae plants resistant to Plasmodiophora brassicae (clubroot)

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- Number: US 10,767,190 B2
- Document ID: US10767190B2
- Status: Granted
- Kind: B2
- Application number: 15/775,580
- Filed: 2016-12-15
- Published: 2019-08-08
- Granted: 2020-09-08
- Earliest claimed: 2015-12-15
- Assignees: BASF Agricultural Solutions Seed US LLC
- Applicants: BASF Agricultural Solutions Seed, US LLC
- Inventors: Steven Engelen; Michel Van Thournout; Stephen Rae; Kim Crommar; Vanessa Hostyn; Godfrey Chongo
- Web page: https://patentgrep.com/patent/US10767190B2

## CPC

- C12N 15/8282: {for fungal resistance}
- A01H 1/045: {using molecular markers}
- C07K 14/415: from plants
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/13: Plant traits
- C12Q 2600/158: Expression markers
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to the identification of clubroot resistance genes from Brassica. Clubroot resistant Brassicaceae plants are provided, as well as clubroot resistance genes and methods and means to increase clubroot resistance in Brassicaceae.

## Claims

### Claim 1

A method for increasing clubroot resistance in a Brassica plant comprising: introducing a CRL clubroot resistance locus in said Brassica plant, and selecting said CRL clubroot resistant Brassica plant for the presence of the CRL clubroot resistance locus by analyzing genomic DNA from said plant for the presence of at least one molecular marker, wherein said at least one molecular marker is linked to the CRL clubroot resistance locus, wherein said CRL clubroot resistance locus comprises the CRL1 and CRL2 clubroot resistance genes, wherein
a) said CRL1 clubroot resistance gene comprises a nucleotide sequence
i) having at least 95% sequence identity to the sequence of nucleotide positions 32750 to 51049 of SEQ ID NO: 1;
ii) having a coding sequence having at least 95% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions 52 to 5340 of SEQ ID NO: 4, nucleotide positions 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; or
ii) encoding a protein having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 11; and
b) said CRL2 clubroot resistance gene comprises a nucleotide sequence having at least 95% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8, or encoding a protein having at least 95% sequence identity to SEQ ID NO: 9.

### Claim 2

A method for producing a clubroot resistant Brassica plant comprising:
introducing a CRL clubroot resistance locus in said Brassica plant, and selecting said CRL clubroot resistant Brassica plant for the presence of the CRL clubroot resistance locus by analyzing genomic DNA from said plant for the presence of at least one molecular marker, wherein said at least one molecular marker is linked to the CRL clubroot resistance locus, wherein said CRL clubroot resistance locus comprises the CRL1 and CRL2 clubroot resistance genes, wherein
a) said CRL1 clubroot resistance gene comprises a nucleotide sequence
i) having at least 95% sequence identity to the sequence of nucleotide positions 32750 to 51049 of SEQ ID NO: 1;
ii) having a coding sequence having at least 95% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions 52 to 5340 of SEQ ID NO: 4, nucleotide positions 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; or
iii) encoding a protein having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 11; and
b) said CRL2 clubroot resistance gene comprises a nucleotide sequence having at least 95% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8; or encoding a protein having at least 95% sequence identity to SEQ ID NO: 9; and
generating progeny from the clubroot resistant Brassica plant comprising the CRL clubroot resistance locus, wherein said progeny is clubroot resistant and comprises said CRL clubroot resistance locus.

### Claim 3 (depends on 2)

The method according to claim 2, wherein said CRL clubroot resistance locus comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1.

### Claim 4

A Brassica plant or plant cell comprising a CRL1 and a CRL2 clubroot resistance gene as transgene, wherein
a) said CRL1 clubroot resistance gene
(i) comprises a coding sequence having at least 95% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions 52 to 5340 of SEQ ID NO: 4, nucleotide positions 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; or (ii) encodes a protein having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 11; and
b) said CRL2 clubroot resistance gene comprises a coding sequence having at least 95% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8, or encodes a protein having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9.

### Claim 5 (depends on 4)

The Brassica plant or plant cell of claim 4, wherein said CRL1 clubroot resistance gene comprises a sequence having at least 95% sequence identity to the sequence of nucleotide positions 32750 to 51049 of SEQ ID NO: 1.

### Claim 6 (depends on 4)

The Brassica plant or plant cell of claim 4, comprising a transgene comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1.

### Claim 7

A method for increasing clubroot resistance in a Brassica plant, comprising:
a) providing a CRL1 and a CRL2 clubroot resistance gene as transgene, b) introducing the CRL1 and CLR2 gene into a Brassica plant cell, to create transgenic cells; and c) regenerating transgenic plants from said transgenic cells; wherein
said CRL1 clubroot resistance gene i) comprises a coding sequence having at least 95% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions 52 to 5340 of SEQ ID NO: 4, nucleotide positions 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; or ii) encodes a protein having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 11; and wherein
said CRL2 clubroot resistance gene comprises a nucleotide sequence having a coding sequence having at least 95% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8, or encodes a protein having at least 95% sequence identity to SEQ ID NO: 9.

### Claim 8 (depends on 2)

A clubroot resistant Brassica plant or plant cell obtainable by the method according to claim 2, said plant or plant cell further comprising at least one other disease resistance gene, said other disease resistance gene is a clubroot resistance gene, a blackleg resistance gene, a Sclerotinia resistance gene, a Verticillium resistance gene, a Fusarium resistance gene, an Aster Yellows resistance gene, an Altemaria resistance gene, or a Grey Stem resistance gene; wherein said other disease resistance gene is a transgene which is genetically linked with said CRL clubroot resistance gene or said clubroot resistance locus.

### Claim 9

A chimeric gene comprising the following operably linked elements:
a) a heterologous plant-expressible promoter;
b) a DNA sequence coding for a CRL1 or for a CRL2 protein; and
c) optionally, a transcription termination and polyadenylation region functional in plant cells, wherein
a) said DNA sequence coding for a CRL1 protein comprises a nucleotide sequence i) having at least 95% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions of 52 to 5340 of SEQ ID NO: 4, nucleotide positions of 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; or ii) encoding a protein having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 11; and
b) said DNA sequence coding for a CRL2 protein comprises a nucleotide sequence having at least 95% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8; or encoding a protein having an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9.

### Claim 10 (depends on 4)

A method to produce clubroot free Brassica plants, comprising sowing seeds from the plants of claim 4, and growing plants from said seeds.

### Claim 11 (depends on 4)

A method of producing food, feed, or an industrial product comprising providing the Brassica plant or plant cell of claim 4, and preparing food, feed or industrial product from the plant or plant cell.

### Claim 12 (depends on 11)

The method of claim 11 wherein
a) the food or feed is oil, meal, grain, starch, flour or protein; or
b) the industrial product is biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said CRL1 clubroot resistance gene comprises a nucleotide sequence
said CRL1 clubroot resistance gene comprises a nucleotide sequence
i) having at least 98% sequence identity to the sequence of nucleotide positions 32750 to 51049 of SEQ ID NO: 1;
ii) having a coding sequence having at least 98% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions 52 to 5340 of SEQ ID NO: 4, nucleotide positions 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; and
iii) encoding a protein having at least 98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 11; and
b) said CRL2 clubroot resistance gene comprises a nucleotide sequence
having a coding sequence having at least 98% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8, or encoding a protein having an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 9.

### Claim 14 (depends on 2)

The method according to claim 2, wherein said CRL clubroot resistance locus comprises a sequence having at least 98% sequence identity to SEQ ID NO: 1.

### Claim 15 (depends on 4)

The Brassica plant or plant cell of claim 4, wherein said CRL1 clubroot resistance gene
i) comprises a coding sequence having at least 98% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions 52 to 5340 of SEQ ID NO: 4, nucleotide positions 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; or
(ii) encodes a protein having an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; and
said CRL.2 clubroot resistance gene
comprises a coding sequence having at least 98% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8;
or encodes a protein having an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 9.

### Claim 16 (depends on 4)

The Brassica plant or plant cell of claim 4, wherein said CRL1 clubroot resistance gene comprises a sequence having at least 98% sequence identity to the sequence of nucleotide positions 32750 to 51049 of SEQ ID NO: 1.

### Claim 17 (depends on 4)

The Brassica plant or plant cell of claim 4, comprising a transgene comprising a sequence having at least 98% sequence identity to SEQ ID NO: 1.

### Claim 18 (depends on 9)

The chimeric gene of claim 9, wherein
said DNA sequence coding for a CRL1 protein comprises a nucleotide sequence
i) having a coding sequence having at least 98% sequence identity to the sequence of nucleotide positions 52 to 5343 of SEQ ID NO: 2, nucleotide positions 52 to 5340 of SEQ ID NO: 4, nucleotide positions 52 to 5361 of SEQ ID NO: 6, or to SEQ ID NO: 10; or
iii) encoding a protein having at least 98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 11; and
b) said DNA sequence coding for CRL2 protein comprises a nucleotide sequence having at least 98% sequence identity to the sequence of nucleotide positions 220 to 2898 of SEQ ID NO: 8, or encoding a protein having at least 98% sequence identity to SEQ ID NO: 9.