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

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

Global Institute for Food Security (GIFS) IPK Gatersleben

Apomixis scientists everywhere thank you to everyone

Apomixis in plants: Naturally occurring asexual reproduction via seeds Sex Parents Apomixis Gametes Offspring

Hybrid breeding in crop plants Inbred line 1 Inbred line 2 Heterosis in the genetically identical 1 st generation hybrids

Cross the F1 generation - Sex shuffles the genome SEX and Meiosis

Apomixis could fix hybrid genotypes in crop plants APOMIXIS

Diversification: the disruptive effects of apomixis Single generation hybrids Production of limitless numbers of hybrid cultivars Niche breeding cultivars tailored to specific environments Farmers become innovators (generate 3 rd world business) Local and Global benefits

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

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 Volume 36, April 2017, Pages 119-128

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 annum 4. If it s so important, why don t we have apomictic crops yet? The propagation-related benefits of apomixis technology could save cassava and potato growers as much as $3.2 billion per year It s complicated

Hieracium crosses did not behave as in Pisum! 1.The F 1 hybrids, which he assumed to be true breeding strains (e.g. Pisum), had high levels of variation. 2.The F 2 generations, on the other hand, were identical, with no expected segregation. Mendel, G., 1869 Ueber einige aus künstlicher Befruchtung gewonnenen Hieracium-Bastarde. Verh. Naturf. Ver. Brünn 8(Abhandlungen): 26 31

Comparative genomics of naturally-occurring apomictic species Rockcress (Boechera) St. Johns wort (Hypericum) Buttercups (Ranunculus) Kentucky bluegrass (Poa)

Apomixis: Sex has been lost!

Sex vs Apomixis: big challenges to finding genetic factors Genome Reproductive fitness Sex DNA repair genetic variability high adaptive potential 2-fold cost heterozygosity (not inbreds) assemble & purge mutations Apomixis low genome plasticity mutation masking structural variations genetically identical progeny fixing of heterozygozity reproductive assurance mutation accumulation Distribution niche construction geographic parthenogenesis pioneers

Arabidopsis, Boechera and Brassica Arabidopsis thaliana Arabidopsis lyrata Boechera sp. Brassica rapa Arabis alpina Cruciferae Arabidopsis tools can be used in Boechera (and we can apply them to Brassica)

Comparing diploid sexuality to diploid apomixis overcomes a common research challenge Diploid sex Diploid and triploid apomixis Sharbel et al.; Cytogenet. Genome Res 2004 Sharbel et al.; Cytogenet. Genome Res 2005

We measure apomixis by analysing fertilization of the central cell (using flow cytometry) APOMIXIS SEX Pollen Embryo sac Embryo sac (2n + 2n) (n + Pollen n) (n + n) OR (n + n) (n + n) OR (2n + 2n) (n) (n) (2n) Embryo? 2n n Endosperm? 3n n 2:4, 2:5, 2:6 etc. Deviations 2 embryo from 2:3 : embryo 3 endosperm : endosperm genome genome ratio ratio

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

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

APOLLO (Apomeiosis Linked-Locus) An apomixis-specific DEDDh exonuclease expressed in premeiotic Boechera ovules Sexual plants (homozygous) SEX SEX Somatic cells Gene expression Ovules Apomictic plants (heterozygous) APO SEX Corral, Vogel, Aliyu, Hensel, Thiel, Kumlehn and Sharbel. Plant Phys (2013)

APOLLO APO allele is highly conserved in ALL apomictic plants

Hybridization and apomixis retrofracta X stricta Sex divaricarpa Apomixis Kantama, L., Sharbel, T.F., H. de Jong et al. (PNAS 2007)

Introduction of APOLLO APO allele induces apomixis Apomict Sexual APOLLO+ APOLLO- Apomictic offspring

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 Sex Sex Sex Sex Glacial Refugium

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 Sex Sex Sex Sex Glacial Refugium

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

APOLLO functional evidence?

Tissue culture, transformation and genome editing 1. Knock out UPGRADE and APOLLO (CRISPR) 2. Introduce APOLLO in a sexual background Boechera sp regenerants (A. Li, Hong Wang; unpublished data)

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.

Major thanks! An Institute of the Leibniz Society 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).