forward genetic screens to identify elements of apomixis ... 27th/afternoon... · apomixis can be...

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Forward Genetic Screens to Identify Elements of Apomixis in Maize Ueli Grossniklaus Institute of Plant Biology of the University of Zürich Zürich-Basel Plant Science Center Apomixis and its Relationship to Sexuality Screen for Apomixis Mutants in Maize

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Page 1: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Forward Genetic Screens to IdentifyElements of Apomixis in Maize

Ueli GrossniklausInstitute of Plant Biology of the University of Zürich

Zürich-Basel Plant Science Center

Apomixis and its Relationship to Sexuality

Screen for Apomixis Mutants in Maize

Page 2: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Apomixis: Asexual Reproductionthrough Seeds

ApomicticProgeny

SexualProgeny

R. BicknellSexualprogeny aregenotypicallyand pheno-typicallydifferent!

Variation asthe basis forplantbreeding!

Apomicticprogeny are

geneticallyand pheno-

typicallyidentical!

Fixation ofhighly

complexgenotypes!

Page 3: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Potential Applications in Agriculture

• Fixation of any heterozygous genotype - however complex -(including F1 hybrids). Facilitates hybrid seed production andwould allow subsistence farmers to save seeds.

Much faster and cheaper breeding programs, also for crops forwhich no or little commercial breeding efforts are made.

Seed propagation of traditional vegetatively propagated crops(vegetative propagation of cassava, potatoes, yams; highlysusceptible to virus infections)

Transgene containment if autonomous apomicts can beobtained and combined with male sterility.

Page 4: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Sexuality and Gametophytic ApomixisAre Closely Related

APOMIXIS

SEXUALITY

UnreducedCell

ReducedGametophyte

No ReductionApomeiosis1)

UnreducedGamteophyte

Embryo Endosperm

Meiosis

DoubleFertilization 2) Partheno-

genesis

Embryo

3)Autonomy orFertilization

Endosperm

Page 5: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Developmental Characteristics ofGametophytic Apomixis

Deregulation of Developmental Processes inSpace and Time (Heterotopy, Heterochrony)

EmbryoSac Initiation

Activation ofEmbryogenesis

EndospermDevelopment

Apomixis Is Controlled by Genes that Play aRole in Sexuality (Sexual Model Systems!)

(1) ApomeiosisPartheno-genesis

FunctionalEndosperm

Grossniklaus et al. (1997) Hybrid Rice; Grossniklaus (2001) Flowering of Apomixis

(2) (3)

Page 6: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Engineering of Apomixis: InducingElements by Misexpression of Candidates

Expression in Egg Gene Inducing Embryo

AutonomousEmbryo

Partheno-genesis

Expression in MMC Gene Avoiding Reduction

Apo-meiosis

UnreducedGametophyte

Grossniklaus (2001) The Flowering of Apomixis

Page 7: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Apomixis Mutants: Exploit theEndosperm

Balance Problemendosperm development in

maize strictly depends on a 2m:1pratio of maternal to paternal

genomes !

2m:1p plumb kernelsdeviations aborted kernels

4m:1p aborted2m:1p normal4m:2p normal

2nx2n 4nx2n

Page 8: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Screen for Mutants Relevant toApomeiosis or Endosperm Formation

Basics: 2n x fertile4n x fertile2n x 4n ; 4n x 2n aborted seeds

A maternal:paternal genome ratio of 2m:1p is required fornormal seed development (imprinting?, dosage effects?,cytoplasmic factors?, nucleo-cytoplasmic ratio?)

Screen: 2n (Muon) x 4nˆR-nj --> if non-reductionmutant (nrm):plump seeds

Page 9: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

X

Generate F2 Families SegregatingMutator (Mu) Induced Mutations

bzmum9

/bzmum9r-g/r-g

X

3200 F2 familiessegregatingnew mutations

F1

+ mut

+ mut

+

mut

+ +

+ mut mut mut

Transposition ofMutator Elements

Page 10: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Identify Families that SegregateMutants Producing Plump Kernels

4n R-nj3200 families with new Mu-induced mutations

X

3 Mutant Classes:

non-reduction mutant(Em 4n, En 6n)

ploidy barrier breakdown (Em 3n, En 4n)

extra polar nuclei(Em 3n, En 6n)

Page 11: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

wild type 2n kernel wild type 4n kernel

aborted kernel mutant nrm2

2c

4c6c

4c 8c

4c8c

3c

Page 12: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Results of Non-Reduction Screen 1~ 1600 families (~ 15-20plants each) x 4nˆR-nj:

4 highly penetrant nrmmutants (2 derived fromsame Mu F1-> nrm2):

• nrm1 (Em 4n, co-dom.)• nrm2 (Em 4n, rec.)• elongate1 (Em 4n, rec.)

(Rhoades & Dempsey, 1966)

Screen 2: nrm3-nrm6• not yet characterized

nrm1/+

nrm1/nrm1

Page 13: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Mechanisms of Production ofUnreduced Embryo Sacs by elongate1• Candidate apomixis gene based on the phenotype (2n

functional embryo sacs) and syntenic position to apomixislocus in Tripascum dactyloides (Savidan, 2000)

But MI appears to be normal based on a half-tetrad analysis(Rhoades & Dempsey 1966):

• MII is skipped• extra S-phase between MI and MII

At the cytogenetic level, el1 chromosomes look less condensedin MI and the recombination frequency is not normal -> is therea problem in MI? (Nel 1979)

Page 14: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

elongate1 skips Meiosis II

wild-typemeiosis

FMS

FMSFMS in elongate1 one of thedyad cells initiates embryosac development(heterochrony!)

Barrell & Grossniklaus (2005) Plant Journal

Page 15: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

nrm1 and nrm2 Show NuclearRestitution Typical of Diplospory

wild type aberrant meioses in nrm2

FMS

nuclearrestitution

Philippa Barrell

Page 16: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

el1 & nrm2 show Aspects of Apomeiosis

MMC

wild type

EmbryoSac

Initiation

Dyad Tetrad

∂∂

FMS

∂∂

∂elongate

∂ ∂∂

FDR

∂nrm2

Diplospory

Page 17: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Screen for Embryos that Developwithout Fertilization:

parthenogenetic (par) Mutants

• Need to be able to grow a normal plant from aputative mutant and propagate it (this is difficult withhaploids)

Need to be able to distinguish normal fertilizationevents from mutants in an straight forward screen

Endosperm development is necessary for viable seedformation (allow pseudogamy)

Page 18: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Basis of the Parthenogenesis Screen• Grow a normal plant from a putative mutant and propagate it: do

screen in el1/el1 --> parthenogenetic offspring is diploid!

Need to be able to distinguish normal fertilization events frommutants: use a dominant paternal marker --> R-nj

Endosperm development is necessary: fertilize with tetra-ploid pollen to allow for sexual endosperm formation!

Page 19: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

el1/el1 ; r1/r1 ; Muon X 4n^R-nj (tetraplex)

~40% to 50% of ovuleshave diploid embryo sacs

The Mutator Parthenogenesis Screen

normal haploid embryo sacs will not produce viableseeds due to the ploidy barrier

most el1/el1 diploid embryo sacs have normal doublefertilization (R-nj embryo and endosperm)

parthenogenic mutants have only fertilization ofcentral cell (r1 embryo and R-nj endosperm)

normal endosperm develop-ment (4m:2p in el1/el1)

Page 20: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

First Results of the MutatorParthenogenesis Screen

Approximately 80’000 kernels were screened forabsence of paternal R-nj marker in the embryo

Putative par* mutants were grown and fertilized with4n^r1 pollen to confirm that the embryos did not arisefrom fertilization and were not carrying R-nj

7 par* mutants recovered and segregatingpopulations are being made for further analyses

Page 21: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

SummaryApomixis can be viewed as a deregulated sexual pathway inspace and time (heterochronic and/or heterotopic expression ofkey regulatory ‘sexual’ genes)

Sexual model systems can be used efficiently to identify keyregulatory genes (lof and gof screens) to engineer apomixis

Maize mutants that produce unreduced embryo sacs (hetero-chrony in el1, nuclear restitution/diplospory in nrm2) havebeen identified, more mutants remain to be characterized

Maize mutants that produce parthenogenetic offspring havebeen identified.

Apomixis can be engineered by combining the different elementsapomeiosis, parthenogenesis, and endosperm adaptations

Page 22: Forward Genetic Screens to Identify Elements of Apomixis ... 27th/Afternoon... · Apomixis can be viewed as a deregulated sexual pathway in space and time (heterochronic and/or heterotopic

Acknowledgements: Apomixis

Gametophytic Genes

R. Baskar *Jay Moore *Amal JohnstonPatrick MeierSamuel Wüst *Anja SchmidtKitty Vijverberg *Olga Kirioukhova

Maize Mutants

Philippa Barrell *Arco BrunnerSharon Kessler

Field Management

Tim Mulligan (CSHL)Mathias Menzi (FAL)

Collaborators

Mark CurtisLukas BrandRichard JeffersonFulvio PupilliHelmut Bäumlein

*former co-workers