# US 2021/0277410 A1 · Transgene and mutational control of sexuality in maize and related grasses

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- Number: US 2021/0277410 A1
- Document ID: US20210277410A1
- Status: Published application
- Kind: A1
- Application number: 16/326,490
- Filed: 2017-08-11
- Published: 2021-09-09
- Earliest claimed: 2016-08-19
- Assignees: Yale University; Rhode Island Council on Postsecondary Education
- Applicants: Yale University; Rhode Island Council On Postsecondary Education
- Inventors: Stephen L. DELLAPORTA; Andrew HAYWARD; Maria MORENO; Albert KAUSCH; John MOTTINGER
- Web page: https://patentgrep.com/patent/US20210277410A1

## CPC

- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/8212: {Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/8287: {for fertility modification, e.g. apomixis}
- C12N 15/8289: {Male sterility}
- C12N 15/829: {Female sterility}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention pertains to genetically modified plants, particularly maize, sorghum and rice, with an all pistillate or all staminate phenotype and methods of the same. The survival of functional pistils in maize requires the action of the sk1 gene. SK1 encodes a glycosyltransferase (GT) that protects pistils from tasselseed-mediated cell death. sk1-dependent pistil protection at a developing floret gives rise to stamen arrest at the same floret, and so determines the pistillate floral fate. This is the first single gain-of-function gene known to control sexuality. The present invention further provides a direct strategy to extend hybrid technologies to related cereals such as sorghum and rice. Tasselseed and silkless genes represent major sex determination genes in maize, a pathway that permits the efficient production of hybrid seed and the associated benefits of heterosis-increased yield, resistance to pathogens, etc. Except for maize, current hybrid systems in cereals are fraught with genetic and environmental limitations. Genotype-independent hybrid cereal technology could potentially increase crop yields as much as 20-40% without placing additional land under agricultural production. This has profound implications for food security and the environmental impact of agriculture in some of the poorest regions of the world.

## Claims

### Claim 1

An isolated polynucleotide encoding a polypeptide of SEQ ID NO: 2 or an amino acid sequence variant thereof operably linked to a heterologous promoter.

### Claim 2 (depends on 1)

The isolated polynucleotide of claim 1, wherein the heterologous promoter is a CaMV 35S promoter.

### Claim 3 (depends on 1)

The isolated polynucleotide of claim 1 further comprising a marker gene.

### Claim 4 (depends on 3)

The isolated polynucleotide of claim 3, wherein the marker gene is an herbicide resistance gene.

### Claim 5 (depends on 4)

The isolated polynucleotide of claim 4, wherein the herbicide resistance gene is bar.

### Claim 6 (depends on 4)

The isolated polynucleotide of claim 4, wherein the herbicide resistance gene encodes 5-enolpyruvyl-shikimate synthase (ESPS).

### Claim 7 (depends on 3)

The isolated polynucleotide of claim 3, wherein the marker gene affects the visual appearance of the seed or seedling.

### Claim 8 (depends on 7)

The isolated polynucleotide of claim 7, wherein the marker gene controls the appearance or distribution of anthrocyanin pigments in the seed or seedling.

### Claim 9 (depends on 1)

A plant cell transformed with the isolated polynucleotide of claim 1.

### Claim 10

A genetically modified plant comprising a transgene containing an sk1-encoded glycosyltransferase operably linked to a promoter for heterologous expression in the cells of the plant.

### Claim 11 (depends on 10)

The genetically modified plant of claim 10, wherein the plant is maize, sorghum or rice.

### Claim 12 (depends on 10)

The genetically modified plant of claim 10, wherein the genetically modified plant is a unisexual plant.

### Claim 13

A genetically modified plant comprising a transgene encoding a uridine diphosphate (UDP) glycosyltransferase.

### Claim 14 (depends on 13)

The genetically modified plant of claim 13, wherein the plant is maize, sorghum or rice.

### Claim 15 (depends on 14)

The genetically modified plant of claim 14, wherein the genetically modified plant comprises inflorescences of the pistillate phenotype associated with sk1.

### Claim 16 (depends on 15)

The genetically modified plant of claim 15, wherein the inflorescences are solely of the pistillate phenotype associated with sk1.

### Claim 17

A genetically modified plant comprising a mutation or transgene targeting an endogenous UDP glycosyltransferase and disrupting its activity.

### Claim 18 (depends on 17)

The plant of claim 17, wherein the UDP glycosyltransferase is sk1.

### Claim 19 (depends on 17)

The genetically modified plant of claim 17, wherein the plant is maize, sorghum or rice.

### Claim 20 (depends on 17)

The genetically modified plant of claim 17 comprising inflorescences of the staminate phenotype associated with the disruption of sk1.

### Claim 21 (depends on 17)

The genetically modified plant of claim 17, wherein the genetically modified plant is a unisexual plant.

### Claim 22 (depends on 17)

The genetically modified plant of claim 17, wherein the mutation is engineered using a CRISPR/Cas9 system.

### Claim 23

A method of generating a genetically modified plant comprising transforming a cell with a construct comprising a transgene encoding a UDP glycosyltransferase, thereby promoting the expression of the UDP glycosyltransferase in one or more cells of the plant.

### Claim 24 (depends on 23)

The method of claim 23, wherein the transgene is sk1.

### Claim 25 (depends on 23)

The method of claim 23, wherein the transgene comprises a polynucleotide encoding a polypeptide of SEQ ID NO: 2 or an amino acid sequence variant thereof.

### Claim 26 (depends on 23)

The method of any one of claim 23, wherein the transgene is operably linked to a heterologous promoter.

### Claim 27 (depends on 26)

The method of claim 26, wherein the heterologous promoter is a CaMV 35S promoter.

### Claim 28 (depends on 23)

The method of claim 23, wherein the UDP glycosyltransferase localizes to a peroxisome.

### Claim 29 (depends on 23)

The method of any one of claim 23, wherein the construct further comprises a marker gene.

### Claim 30 (depends on 29)

The method of claim 29, wherein the marker gene is an herbicide resistance gene.

### Claim 31 (depends on 30)

The method of claim 30, wherein the herbicide resistance gene is bar.

### Claim 32 (depends on 30)

The method of claim 30, wherein the herbicide resistance gene encodes 5-enolpyruvyl-shikimate synthase (ESPS).

### Claim 33 (depends on 29)

The method of claim 29, wherein the marker gene affects the visual appearance of a seed or seedling.

### Claim 34 (depends on 33)

The method of claim 33, wherein the marker gene controls the appearance or distribution of one or more anthrocyanin pigments in the seed or seedling.

### Claim 35 (depends on 29)

The method according to claim 29, further comprising using the marker gene to select at least one genetically modified plant.

### Claim 36 (depends on 35)

The method according to claim 35, further comprising using the genetically modified plant to generate a hybrid seed.

### Claim 37 (depends on 29)

The method according to claim 29, wherein the plant is maize, rice or sorghum.

### Claim 38

A method of generating a transgenic plant comprising the step of engineering a mutation or transgene targeting an endogenous UDP glycosyltransferase and disrupting its activity.

### Claim 39 (depends on 38)

The method of claim 38, wherein the UDP glycosyltransferase is sk1.

### Claim 40 (depends on 38)

The method of claim 38, wherein the plant is maize, sorghum or rice.

### Claim 41 (depends on 38)

The method of claim 38, wherein the plant comprises at least one inflorescence of the staminate phenotype associated with the disruption of sk1.

### Claim 42 (depends on 40)

The method of claim 40, wherein the wherein the transgenic plant is a unisexual plant.

### Claim 43 (depends on 38)

The method of claim 38, wherein the mutation is engineered using a CRISPR/Cas9 system.

### Claim 44

A method of generating a transgenic plant comprising engineering a mutation in a 5′ or 3′ regulatory element of an endogenous UDP glycosyltransferase to alter an expression level of the UDP glycosyltransferase.

### Claim 45 (depends on 44)

The method of claim 44, wherein the transgenic plant is maize, rice or sorghum.

### Claim 46 (depends on 44)

The method of claim 44, wherein the transgenic plant is a unisexual plant.

### Claim 47 (depends on 44)

The method of any one of claim 44, wherein the mutation is engineered using a crispr/Cas9 system, zinc-finger nucleases or transcription activator-like effects.