# US 2014/0157448 A1 · Cichorium spp. male sterile mutants

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- Number: US 2014/0157448 A1
- Document ID: US20140157448A1
- Status: Published application
- Kind: A1
- Application number: 14/091,051
- Filed: 2013-11-26
- Published: 2014-06-05
- Earliest claimed: 2011-05-27
- Assignees: AZ. Agricola T.&T. Produce Di Tiozzo Silvano
- Applicants: AZ. Agricola T.&T. Produce Di Tiozzo Silvano
- Inventors: GIANNI BARCACCIA; Silvano Caenazzo Tiozzo
- Web page: https://patentgrep.com/patent/US20140157448A1

## CPC

- C12Q 1/6895: for plants, fungi or algae
- A01H 1/04: Processes of selection {involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection}
- A01H 5/02: Flowers
- A01H 6/14: Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
- C12N 15/8289: {Male sterility}
- 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
- 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)

## Abstract

The present invention relates to leaf chicory (Cichorium intybus subsp. intybus var. foliosum) nuclear recessive male sterile mutants, to a newly identified polymorphic molecular marker for the nuclear recessive male sterile character in this species, to methods for the selection of leaf chicory nuclear recessive male sterile mutants, to methods for the production of seed parent male sterile and pollen donor male fertile inbred lines of leaf chicory, including all cultivated types of radicchio, that are, respectively, homozygous for the nuclear recessive male sterile mutant allele or homozygous for the male fertile wildtype allele, and to methods for the constitution of F1 hybrids that are all heterozygous at the ms locus.

## Claims

### Claim 1

A male sterile mutant of leaf chicory, including Radicchio (Cichorium intybus subsp. intybus var. foliosum), parts or derivatives thereof, wherein the male sterility trait: 1) shows a cytological phenotype characterized by shapeless, small and shrunken microspores in dehiscent anthers, that arrest their development at the uninucleate stage, collapsing before their release from the tetrads; and 2) is controlled by a nuclear recessive mutation (ms) ascribable to a single gene linked 5.8 cM apart to a polymorphic molecular marker locus finely mapped on linkage group 4 comprising a microsatellite or simple sequence repeat (TC)n in SEQ. ID NO 1 or 2 and the progeny thereof, wherein progeny plants may show either a mutant or a wild-type cytological phenotype for said trait and may be at least heterozygous for said mutation at the ms locus.

### Claim 2 (depends on 1)

The plant of claim 1 wherein said plant belong to Cichorium intybus subsp. intybus var. foliosum.

### Claim 3 (depends on 1)

The plant of claim 1 wherein n of said (TC)n is 27 or 28 (SEQ ID NO: 12).

### Claim 4

A polymorphic genetic marker comprising SEQ ID NO 1, said marker being linked to a locus of nuclear recessive male sterile mutation (ms) in leaf chicory species, including all biotypes of cultivated Radicchio.

### Claim 5 (depends on 4)

The marker of claim 4 wherein said marker comprises SEQ ID NO 2.

### Claim 6 (depends on 4)

The marker of claim 4 wherein the linkage thereof to the locus of said nuclear recessive mutation (ms) is such that a recombination frequency of about 5.8% is observed between the two loci.

### Claim 7

A method for the selection of mutant plants of leaf chicory, including all Radicchio cultivated types, that are homozygous or heterozygous for a mutation inducing nuclear recessive male sterility (ms) linked to a polymorphic genetic marker comprising a simple sequence repeat (TC)n in SEQ ID NO 1 or 2 comprising the steps of:
genotyping the leaf chicory plants or parts thereof by analysing their DNA for the simple sequence repeat (TC)n in SEQ ID NO 1 or SEQ ID NO 2;
comparing the genotypes thus obtained to a male sterile genotype of reference (msms) for said simple sequence repeat (TC)n target DNA region; and
selecting the plants having at least one marker allele of said sequence repeat where n is equal to the n of said male sterile genotype of reference.

### Claim 8 (depends on 7)

The method of claim 7 wherein said plants belong to leaf chicory (Cichorium intybus subsp. intybus var. foliosum), including all cultivated types of Radicchio.

### Claim 9 (depends on 7)

The method of claim 7 wherein said thus obtained genotype comprises at least one simple sequence repeat (TC)n in SEQ ID NO 1 or 2 wherein n is 27 or 28 (SEQ ID NO: 12).

### Claim 10

A method for the production of mutant male sterile seed parents of leaf chicory plant materials wherein the male sterility is a nuclear recessive male sterility due to mutation of a nuclear gene (ms) linked to a polymorphic genetic marker comprising a simple sequence repeat (TC)n in SEQ ID NO 1 or 2, comprising the steps of:
genotyping the leaf chicory plants or parts thereof by analysing their DNA for the simple sequence repeat (TC)n in SEQ ID NO 1 or SEQ ID NO 2,
comparing the genotypes thus obtained to a male-sterile genotype of reference (msms) for said simple sequence repeat (TC)n target DNA region; and
selecting the plants having both marker alleles of said sequence repeat where n is equal to the n of said male sterile genotype of reference.

### Claim 11 (depends on 10)

The method of claim 10 further comprising the steps of
selecting plants having at least one marker allele for said sequence repeat wherein n is equal to the n of said male sterile genotype of reference,
submitting said plants to hybridization and selection for other traits, repeating said genotyping on said plants and comparing the genotype thus obtained to said genotype of reference, before the step of
selecting the plants having both alleles of said repeat where n is equal to the n of said male sterile genotype of reference.

### Claim 12 (depends on 10)

The method of claim 10 wherein said plants belong to leaf chicory (Cichorium intybus subsp. intybus var. foliosum), including all cultivated types of Radicchio.

### Claim 13 (depends on 10)

The method of claim 10 wherein said thus obtained genotype comprises only Simple Sequence Repeat (TC)n in SEQ ID NO 1 or 2 wherein n is 27 or 28 (SEQ ID NO: 12).

### Claim 14

A method for the production of mutant F1 hybrids of leaf chicory (Cichorium intybus subsp. intybus var. foliosum), including all cultivated types of Radicchio wherein said hybrids are fertile and heterozygous for a mutation in a gene inducing nuclear recessive male sterility (ms) linked to a polymorphic genetic marker comprising a simple sequence repeat (TC)n in SEQ ID NO 1 or 2 comprising the steps of:
genotyping the leaf chicory plants or parts thereof by analysing their DNA for the simple sequence repeat (TC)n in SEQ ID NO 1 or SEQ ID NO 2,
comparing the genotypes thus obtained to a male-sterile genotype of reference (msms) for said simple sequence repeat (TC)n,
selecting the plants having both marker alleles of said sequence repeat where n is equal to the n of said male sterile genotype of reference thus obtaining male sterile (msms) seed parent plants,
selecting plants having both marker alleles of said sequence repeat where n is different from the n of said male sterile genotype of reference thus obtaining male fertile (MsMs) pollen donor plants, and
crossing said seed parent plants with said pollen donor plants and collecting the highly heterozygous F1 seed thus obtained.

### Claim 15 (depends on 14)

The method of claim 14 further comprising the steps of
selecting plants having at least one marker allele for said sequence repeat wherein n is equal to the n of said male sterile genotype of reference,
submitting said plants to hybridization and selection for other traits, repeating said genotyping on said plants and comparing the genotype thus obtained to said genotype of reference, before the steps of
selecting the plants having both marker alleles of said sequence repeat where n is equal to the n of said male sterile genotype of reference thus obtaining male sterile (msms) seed parent plants,
selecting plants having both marker alleles of said sequence repeat where n is different from the n of said male sterile genotype of reference thus obtaining male fertile (MsMs) pollen donor plants, and
crossing said seed parent plants with said pollen donor plants and collecting the F1 seed thus obtained.

### Claim 16 (depends on 14)

The method of claim 14 wherein said plants comprise leaf chicory (Cichorium intybus subsp. intybus var. foliosum).

### Claim 17 (depends on 14)

The method of claim 14 wherein said genotype of said male sterile seed parent plants comprises only simple sequence repeats (TC)n in SEQ ID NO 1 or 2 in wherein n is 27 or 28 (SEQ ID NO: 12) and the genotype of said pollen donor plants comprises simple sequence repeats (TC)n in SEQ ID NO 1 or 2 wherein n is different from 27 or 28 (SEQ ID NO: 12).

### Claim 18 (depends on 7)

Plants or parts or elaborates thereof selected according to the methods of claim 7.

### Claim 19 (depends on 7)

A kit for carrying out the methods of claim 7 comprising at least one aliquot of oligonucleotides and optionally reagents for the genotyping analyses based on the simple sequence repeat (TC)n markers shown in SEQ ID NO 1 or in SEQ ID NO 2.

### Claim 20

A male sterile mutant of Cichorium intybus subsp. intybus var. foliosum, parts or derivatives thereof, wherein the genotype responsible for male sterility is ascribable to a recessive mutation (ms) of a single nuclear gene tightly linked to a polymorphic molecular marker comprising a microsatellite or simple sequence repeat (SSR) that is a (TC)n repetition, wherein n is an integer ranging between 27 and 33 (SEQ ID NO: 7), mapped on the linkage group 4 of the Cichorium intybus genome consensus map and characterized in that said microsatellite marker shows genetic co-segregation with the ms mutant gene, being the genetic distance estimated between the two corresponding loci of about 6 cM.

### Claim 21 (depends on 20)

A male sterile Cichorium intybus subsp. intybus var. foliosum mutant according to claim 20, wherein said ms mutants show a phenotype of dehiscent anthers with no pollen grains and wherein different forms of meiotic abnormalities occur, such as partial or aspecific pairing between homologous chromosomes at prophase I and several chromatin bridges at anaphase and telophase II, resulting in 100% of non-viable microspores that arrest their development at the uninucleate stage when they appear shapeless, shrunken and smaller than wild-type microspores, collapsing before their release from the tetrads.

### Claim 22 (depends on 20)

A male sterile Cichorium intybus subsp. intybus var. foliosum mutant according to claim 20, wherein the marker comprising SEQ ID NO 1 or consisting of SEQ ID NO 2, is E02M09/230.

### Claim 23 (depends on 20)

A male sterile Cichorium intybus subsp. intybus var. foliosum mutant according to claim 20, wherein the number of (TC)n repeats in the said microsatellite marker is equal to 27 or 28 (SEQ ID NO: 12).

### Claim 24

A polymorphic molecular marker comprising SEQ ID NO 1 or consisting of SEQ ID NO 2 that is a (TC)n repetition wherein n is an integer ranging from 25 up to 39 (SEQ ID NO: 13), said marker being genetically linked to a locus of nuclear recessive male sterile mutation (ms) in Cichorium intybus subsp. intybus var. foliosum characterized in that the microsatellite marker shows a mean recombination frequency with the ms mutation gene of about 5.8% so that the two corresponding loci are at a relative genetic distance of about 6 cM.

### Claim 25 (depends on 24)

The marker of claim 24 wherein said marker comprises SEQ ID NO 1 or consists of SEQ ID NO 2, including the microsatellite (TC) repeat.

### Claim 26

A male sterile mutant of a plant, parts or derivatives thereof, wherein the male sterility trait is controlled by a nuclear recessive mutation (ms).

### Claim 27 (depends on 26)

The plant of claim 26, wherein the male sterility trait shows a cytological phenotype characterized by shapeless, small and shrunken microspores in dehiscent anthers, that arrest their development at the uninucleate stage, collapsing before their release from the tetrads.

### Claim 28 (depends on 26)

The plant of claim 26, wherein the nuclear recessive mutation (ms) is ascribable to a single gene linked 5.8 cM apart to a polymorphic molecular marker locus finely mapped on linkage group 4 comprising a microsatellite or simple sequence repeat (TC)n in SEQ. ID NO 1 or 2 and the progeny thereof.

### Claim 29 (depends on 28)

The plant of claim 28, wherein progeny plants may show either a mutant or a wild-type cytological phenotype for said trait and may be at least heterozygous for said mutation at the ms locus.

### Claim 30 (depends on 26)

F1 hybrid plants or parts or elaborates thereof obtained using the plant of claim 26.

### Claim 31 (depends on 30)

Segregant progeny plants or parts or elaborates thereof obtained using the F1 hybrid plants of claim 30.