# US 10,767,189 B2 · Methods for producing cotton plants with enhanced drought tolerance and compositions thereof

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- Number: US 10,767,189 B2
- Document ID: US10767189B2
- Status: Granted
- Kind: B2
- Application number: 15/237,136
- Filed: 2016-08-15
- Published: 2017-02-23
- Granted: 2020-09-08
- Earliest claimed: 2015-08-18
- Assignees: Monsanto Technology LLC
- Applicants: Monsanto Technology LLC
- Inventors: Issa Coulibaly; Nilesh Dighe; Xuehui Feng; Richard H. Sheetz; Humphrey W. Wanjugi
- Web page: https://patentgrep.com/patent/US10767189B2

## CPC

- C12N 15/8273: {for drought, cold, salt resistance}
- A01H 1/045: {using molecular markers}
- A01H 5/10: Seeds
- A01H 6/604: {Gossypium [cotton]}
- 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
- 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

## Abstract

The present disclosure is in the field of plant breeding. The disclosure provides methods for breeding cotton plants having a staygreen trait using marker-assisted selection. The disclosure further provides germplasm that stays green under high drought stress and provide yield advantage over non-staygreen germplasm. The disclosure also provides genetic markers associated with staygreen QTLs for introgressing these QTLs into elite germplasm in a breeding program and producing novel cotton germplasm that performs better under water-limited conditions.

## Claims

### Claim 1

A method of creating a population of cotton plants or seeds, said method comprising the steps of:
a. identifying in a first population of staygreen (STG) cotton plants or seeds, cotton plants having at least one marker linked within 3.0 cM to at least one haplotype comprising three or more representative STG alleles in at least one polymorphic locus;
b. selecting from said first population one or more cotton plants or seeds comprising said at least one marker linked to said at least one haplotype comprising three or more representative STG alleles; and
c. crossing said selected one or more cotton plants or seeds to produce a second population of cotton plants or seeds comprising an STG phenotype and said at least one haplotype comprising three or more representative STG alleles;
wherein said haplotype comprises three representative STG alleles of the haplotype of quantitative trail locus (QTL) 1 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:1,
an A nucleotide at position 61 of SEQ ID NO:2,
an A nucleotide at position 61 of SEQ ID NO:3,
a T nucleotide at position 440 of SEQ ID NO:4,
a C nucleotide at position 61 of SEQ ID NO:5,
an A nucleotide at position 61 of SEQ ID NO:6, and
an A nucleotide at position 61 of SEQ ID NO:7,
the haplotype of QTL 2 selected from the group consisting of: an A nucleotide at position 61 of SEQ ID NO:8,
a G nucleotide at position 61 of SEQ ID NO:9,
a G nucleotide at position 61 of SEQ ID NO:10,
an insertion beginning at position 224 of SEQ ID NO:11,
an A nucleotide at position 61 of SEQ ID NO:12,
a G nucleotide at position 61 of SEQ ID NO:13,
a G nucleotide at position 61 of SEQ ID NO:14,
a C nucleotide at position 61 of SEQ ID NO:15,
a T nucleotide at position 292 of SEQ ID NO:16, and
a C nucleotide at position 104 of SEQ ID NO:17;
the haplotype of QTL 3 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:18,
a C nucleotide at position 61 of SEQ ID NO:19,
a G nucleotide at position 50 of SEQ ID NO:20, and
an A nucleotide at position 61 of SEQ ID NO:21;
the haplotype of QTL 4 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:22,
a G nucleotide at position 61 of SEQ ID NO:23,
a T nucleotide at position 230 of SEQ ID NO:24,
a G nucleotide at position 61 of SEQ ID NO:25,
an A nucleotide at position 61 of SEQ ID NO:26,
a C nucleotide at position 150 of SEQ ID NO:27, and
an A nucleotide at position 391 of SEQ ID NO:28;
the haplotype of QTL 5 selected from the group consisting of:
a C nucleotide at position 61 of SEQ ID NO:29,
a C nucleotide at position 61 of SEQ ID NO:30,
a T nucleotide at position 61 of SEQ ID NO:31,
and an A nucleotide at position 230 of SEQ ID NO:32;
the haplotype of QTL 6 selected from the group consisting of:
a C nucleotide at position 61 of SEQ ID NO:33,
a T nucleotide at position 61 of SEQ ID NO:34,
a G nucleotide at position 61 of SEQ ID NO:35,
a T nucleotide at position 156 of SEQ ID NO:36,
an A nucleotide at position 61 of SEQ ID NO:37,
a T nucleotide at position 61 of SEQ ID NO:38,
a C nucleotide at position 61 of SEQ ID NO:39, and
a C nucleotide at position 61 of SEQ ID NO:40; or
the haplotype of QTL 7 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:41;
an A nucleotide at position 61 of SEQ ID NO:42,
an A nucleotide at position 61 of SEQ ID NO:43; and
a C nucleotide at position 61 of SEQ ID NO:44.

### Claim 2 (depends on 1)

The method of claim 1, wherein said at least one marker is located in a chromosomal haplotype flanked by:
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 1 to 7;
any two polymorphic loci elected from the group consisting of SEQ ID NOs: 8 to 17;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 18 to 21;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 22 to 28;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 29 to 32;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 33 to 40,
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 41 to 44, or combinations thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein said at least one marker is within said at least one haplotype comprising three or more representative STG alleles.

### Claim 4 (depends on 1)

The method of claim 1, wherein said at least one haplotype comprising three or more representative STG alleles confers no seed or fiber yield penalty under water-sufficient conditions.

### Claim 5 (depends on 4)

The method of claim 4, wherein the seed or fiber yield under water-limited conditions of cotton plants comprising said representative STG allele is 3% or more higher than the seed or fiber yield of cotton plants without said at least one haplotype comprising three or more representative STG alleles under water-limited conditions.

### Claim 6 (depends on 1)

The method of claim 1, further comprising producing said first population of cotton plants or seeds by crossing a first cotton plant comprising at least one haplotype comprising three or more representative STG alleles, with a second cotton plant that does not comprise said at least one haplotype comprising three or more representative STG alleles of said first cotton plant.

### Claim 7 (depends on 4)

The method of claim 4, wherein said at least one haplotype comprising three or more representative STG alleles has no impact in said second population of cotton plants or seeds over at least one or more traits at maturity selected from the group consisting of plant height, micronaire, bolls open percentage, fiber elongation percentage, fiber maturity ratio, short fiber content, fiber length, fiber strength, uniformity index, lint percentage, and stringout rating, under either water-limited conditions or water-sufficient conditions.

### Claim 8 (depends on 1)

The method of claim 1, wherein said method comprises selecting cotton plants or seeds for at least two markers linked within 3.0 cM to at least two different haplotypes comprising three or more representative STG alleles loci.

### Claim 9 (depends on 8)

The method of claim 8, wherein at least three markers are linked with at least three different STG QTL haplotypes selected from the group consisting of STG QTL haplotypes 1 to 7.

### Claim 10

A method for selecting a cotton plant or seed comprising a representative staygreen (STG) allele, said method comprising:
a. crossing an STG cotton plant, comprising at least one haplotype comprising three or more representative STG alleles, to a second cotton plant lacking said haplotype;
b. detecting a marker linked within 3.0 centimorgan (cM) of said at least one haplotype comprising three or more representative STG alleles in a progeny cotton plant produced from said cross; and
c. selecting said progeny cotton plant or seed comprising said haplotype comprising three or more representative STG alleles;
wherein said haplotype comprises three representative STG alleles of the haplotype of quantitative trail locus (QTL) 1 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:1,
an A nucleotide at position 61 of SEQ ID NO:2,
an A nucleotide at position 61 of SEQ ID NO:3,
a T nucleotide at position 440 of SEQ ID NO:4,
a C nucleotide at position 61 of SEQ ID NO:5,
an A nucleotide at position 61 of SEQ ID NO:6, and
an A nucleotide at position 61 of SEQ ID NO:7,
the haplotype of QTL 2 selected from the group consisting of: an A nucleotide at position 61 of SEQ ID NO:8,
a G nucleotide at position 61 of SEQ ID NO:9,
a G nucleotide at position 61 of SEQ ID NO:10,
an insertion beginning at position 224 of SEQ ID NO:11,
an A nucleotide at position 61 of SEQ ID NO:12,
a G nucleotide at position 61 of SEQ ID NO:13,
a G nucleotide at position 61 of SEQ ID NO:14,
a C nucleotide at position 61 of SEQ ID NO:15,
a T nucleotide at position 292 of SEQ ID NO:16, and
a C nucleotide at position 104 of SEQ ID NO:17;
the haplotype of QTL 3 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:18,
a C nucleotide at position 61 of SEQ ID NO:19,
a G nucleotide at position 50 of SEQ ID NO:20, and
an A nucleotide at position 61 of SEQ ID NO:21;
the haplotype of QTL 4 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:22,
a G nucleotide at position 61 of SEQ ID NO:23,
a T nucleotide at position 230 of SEQ ID NO:24,
a G nucleotide at position 61 of SEQ ID NO:25,
an A nucleotide at position 61 of SEQ ID NO:26,
a C nucleotide at position 150 of SEQ ID NO:27, and
an A nucleotide at position 391 of SEQ ID NO:28;
the haplotype of QTL 5 selected from the group consisting of:
a C nucleotide at position 61 of SEQ ID NO:29,
a C nucleotide at position 61 of SEQ ID NO:30,
a T nucleotide at position 61 of SEQ ID NO:31,
and an A nucleotide at position 230 of SEQ ID NO:32;
the haplotype of QTL 6 selected from the group consisting of:
a C nucleotide at position 61 of SEQ ID NO:33,
a T nucleotide at position 61 of SEQ ID NO:34,
a G nucleotide at position 61 of SEQ ID NO:35,
a T nucleotide at position 156 of SEQ ID NO:36,
an A nucleotide at position 61 of SEQ ID NO:37,
a T nucleotide at position 61 of SEQ ID NO:38,
a C nucleotide at position 61 of SEQ ID NO:39, and
a C nucleotide at position 61 of SEQ ID NO:40; or
the haplotype of QTL 7 selected from the group consisting of:
an A nucleotide at position 61 of SEQ ID NO:41;
an A nucleotide at position 61 of SEQ ID NO:42,
an A nucleotide at position 61 of SEQ ID NO:43; and
a C nucleotide at position 61 of SEQ ID NO:44.

### Claim 11 (depends on 10)

The method of claim 10, wherein said marker is located in a chromosomal haplotype flanked by:
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 1 to 7;
any two polymorphic loci elected from the group consisting of SEQ ID NOs: 8 to 17;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 18 to 21;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 22 to 28;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 29 to 32;
any two polymorphic loci selected from the group consisting of SEQ ID NOs: 33 to 40, or any two polymorphic loci selected from the group consisting of SEQ ID NOs: 41 to 44.

### Claim 12 (depends on 1)

The method of claim 1, wherein said at least one marker is within 1 cM of said haplotype comprising three or more representative STG alleles.

### Claim 13 (depends on 10)

The method of claim 10, wherein said marker is within 1 cM of said haplotype comprising three or more representative STG alleles.

### Claim 14 (depends on 2)

The method of claim 2, wherein said any two polymorphic loci are SEQ ID NOs: 4 and 5.

### Claim 15 (depends on 2)

The method of claim 2, wherein said any two polymorphic loci are SEQ ID NOs: 16 and 17.

### Claim 16 (depends on 1)

The method of claim 1, further comprising producing said first population of cotton plants or seeds by crossing a first cotton plant comprising at least one haplotype comprising three or more representative STG alleles linked with a polymorphic locus selected from the group consisting of SEQ ID NOs: 1 to 44, with a second cotton plant comprising a different at least one haplotype comprising three or more representative STG alleles linked with a polymorphic locus selected from the group consisting of SEQ ID NOs: 1 to 44; wherein each said polymorphic locus is linked with a different STG QTL haplotype selected from the group consisting of STG QTL haplotypes 1 to 7.

### Claim 17 (depends on 16)

The method of claim 16, wherein said second cotton plant comprises at least two different haplotypes comprising three or more representative STG alleles in at least two polymorphic loci selected from the group consisting of SEQ ID NOs: 1 to 44, wherein said polymorphic loci are linked with at least two different STG QTL haplotypes selected from the group consisting of STG QTL haplotypes 1 to 7.

### Claim 18 (depends on 16)

The method of claim 16, wherein said second cotton plant comprises at least three different haplotypes comprising three or more a representative STG allele in at least three polymorphic loci selected from the group consisting of SEQ ID NOs: 1 to 44, wherein said polymorphic loci are linked with at least three different STG QTL haplotypes selected from the group consisting of STG QTL haplotypes 1 to 7.