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