Fixing complex genotypes in plants: progress with apomixis in the Brassicaceae

IPK Gatersleben

Global Institute for Food Security (GIFS)

Apomixis scientists everywhere – thank you to everyone

Gametes

Parents

Apomixis in plants: Naturally occurring asexual reproduction via seeds

Offspring

Sex Apomixis

Inbred line 1 Inbred line 2

Heterosis in the genetically identical 1st generation hybrids

Hybrid breeding in crop plants

Cross the F1 generation - Sex shuffles the genome

SEX and Meiosis

APOMIXIS

Apomixis could fix hybrid genotypes in crop plants

• Single generation hybrids

• Production of limitless numbers of hybrid cultivars

• Niche breeding cultivars tailored to specific environments

• Farmers become innovators (generate 3rd world business)

• Local and Global benefits

Diversification: the disruptive effects of apomixis

Genomic selection: exploiting genetic and phenotypic diversity

Heterosis: male sterility, tester lines, general and specific combining abilities…

• Time and resources • Only a small fraction of diversity is available

Volume 36, April 2017, Pages 119-128

Brassicaceae 1. Combine genomes (natural

crosses, somatic fusion, embryo rescue etc.)

2. Choose and test the best phenotype

3. Turn on apomixis and go immediately to seed increase

“The stabilization of heterozygous genotypes using apomixis would make breeding programs faster and cheaper resulting in an estimated benefit, in the production of hybrid rice alone, of more than $2.5 billion per annum4.”

“The propagation-related benefits of apomixis technology could save cassava and potato growers as much as $3.2 billion per year…”

If it’s so important, why don’t we have apomictic crops yet? It’s complicated…

Mendel, G., 1869 Ueber einige aus künstlicher Befruchtung gewonnenen Hieracium-Bastarde. Verh. Naturf. Ver. Brünn 8(Abhandlungen): 26–31

Hieracium crosses did not behave as in Pisum!

1.The F1 hybrids, which he assumed to be “true breeding” strains (e.g. Pisum), had high levels of variation.

2.The “F2 generations”, on the other hand, were identical, with no expected segregation.

Comparative genomics of naturally-occurring apomictic species

Rockcress (Boechera) St. Johns wort

(Hypericum)

Buttercups (Ranunculus) Kentucky bluegrass

(Poa)

Apomixis: Sex has been lost!

Reproductive fitness

• genetically identical progeny • fixing of heterozygozity • reproductive assurance • mutation accumulation

Genome

Distribution

• low genome plasticity • mutation masking • structural variations

• geographic parthenogenesis • pioneers

Sex Apomixis

Sex vs Apomixis: big challenges to finding genetic factors

• DNA repair • genetic variability

• high adaptive potential • „2-fold cost“ • heterozygosity (not inbreds) • assemble & purge mutations

• niche construction

Arabidopsis, Boechera and Brassica

Arabidopsis tools can be used in Boechera (and we can apply them to Brassica)

Arabidopsis thaliana Arabidopsis

lyrata

Boechera sp.

Brassica rapa

Arabis alpina

Cruciferae

Comparing diploid sexuality to diploid apomixis overcomes a common research challenge

Sharbel et al.; Cytogenet. Genome Res 2004 Sharbel et al.; Cytogenet. Genome Res 2005

Diploid sex

Diploid and triploid apomixis

We measure apomixis by analysing fertilization of the central cell (using flow cytometry)

Embryo 2n

Endosperm 3n

2 embryo : 3 endosperm genome ratio

SEX

Pollen (n + n)

Embryo sac

(n + n)

(n)

(n + n)

Embryo ? n

Endosperm ? n

Deviations from 2:3 embryo : endosperm genome ratio

APOMIXIS

(n + n)

(n)

(2n + 2n)

Pollen Embryo sac

(2n + 2n)

(2n)

OR

2:4, 2:5, 2:6 etc.

OR

Aliyu, Schranz and Sharbel. 2010. Am. J. Botany 97(10): 1719-1731.

Gene expression

Quantifying naturally-occurring phenotypic variation in reproductive traits

Boechera: Genotype-specific variation for apomeiosis expression (71 accessions, 3 individual per accession, >22 000 single seeds)

Megaspore Mother

Cell (MMC)

Quantifying variability in genetic factors underlying favorable reproductive traits • Live tissue microdissections are routine (anthers, pollen, endosperm, etc)

APOLLO (Apomeiosis Linked-Locus) An apomixis-specific DEDDh exonuclease expressed in premeiotic Boechera ovules

Corral, Vogel, Aliyu, Hensel, Thiel, Kumlehn and Sharbel. Plant Phys (2013)

APO

SEX

SEX

SEX

Sexual plants (homozygous)

Apomictic plants (heterozygous)

Gene expression

Somatic cells Ovules

APOLLO APO allele is highly conserved in ALL apomictic plants

retrofracta

X

stricta

divaricarpa

Sex

Apomixis

Kantama, L., Sharbel, T.F., H. de Jong et al. (PNAS 2007)

Hybridization and apomixis

APOLLO+

Apomict

APOLLO-

Sexual

Apomictic offspring

Introduction of APOLLO APO allele induces apomixis

APOLLO - evidence for “contagious” apomixis

Mau M, Lovell JT, Corral JM, Kiefer C, Koch MA, Aliyu OM, Sharbel TF; 2015 PNAS

Post-Pleistocene colonization of NA

Glacial Refugium

Sex

Sex

Sex Sex

APOLLO - evidence for “contagious” apomixis

Mau M, Lovell JT, Corral JM, Kiefer C, Koch MA, Aliyu OM, Sharbel TF; 2015 PNAS

Post-Pleistocene colonization of NA

Glacial Refugium

Sex

Sex

Sex Sex

Post-Pleistocene colonization of NA followed by introgression of APOLLO allele

APOLLO

Sex-Apo

Sex-Apo

Sex-Apo Sex-Apo

APOLLO functional evidence?

Tissue culture, transformation and genome editing

Boechera sp regenerants (A. Li, Hong Wang; unpublished data)

1. Knock out UPGRADE and APOLLO (CRISPR) 2. Introduce APOLLO in a sexual background

Protein Interaction Maps in sexual and apomictic Boechera

APO-allele of APOLLO – High incidence of interactions with particular transcription factors

1. Fully assembled and polished sexual genome 2. 6 apomictic genomes. 3. Sex- versus Apomixis-specific mRNA isoforms in developing ovules (Boechera)

Sexual and apomictic Boechera genomes: a collaboration with NRGene and Gydle

Summary 1. Sexual speciation preceded the evolution of apomixis.

2. Apomixis induced repeatedly in different sexual genetic backgrounds and

tightly linked to the APOLLO-APO allele.

In progress: 1. Functional analyses of APOLLO in Maize and Brassica

2. 3 high-quality apomictic genomes (we’ve completed a sexual reference).

3. Differentially spliced mRNA spectrum of sexual/apomictic ovules.

4. Protein-protein, protein-DNA interactions of sexual/apomictic ovules.

There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved.

— Charles Darwin Concluding remarks in final chapter, The Origin of Species (1859).

Major thanks!

An Institute of the Leibniz Society