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Emergence of novel phenotypes in co-evolving biological systems: allelic diversification and dominance at the Self-incompatibility locus in Arabidopsis.

Periodic Reporting for period 4 - NOVEL (Emergence of novel phenotypes in co-evolving biological systems: allelic diversification and dominance at the Self-incompatibility locus in Arabidopsis.)

Reporting period: 2020-04-01 to 2021-05-31

• What is the problem/issue being addressed?
For most biological systems, establishing the link between genotype and phenotype and between phenotype and fitness is a challenge task. In this project, we focus on the sporophytic self-incompatibility system in outcrossing Arabidopsis species, a model biological system in which two distinct co-evolutionary processes are becoming well-understood: 1) between the male and female reproductive proteins allowing self-pollen recognition and rejection and 2) between small non-coding RNAs and their target sites that jointly control the dominance/recessivity interactions between self-incompatibility alleles. By studying these two model systems, we improve our understanding of how functional and regulatory novelty can arise in natural populations.

• Why is it important for society?
The NOVEL project has improved basic knowledge by developing an integrated understanding of the proximal causes of phenotypic variation (at the mechanistic level) but also of the ultimate causes (in terms of how natural selection is acting on those variations). This is important in order to improve the predictive power of evolutionary biology by clarifying the molecular constraints on the evolutionary process. At the applied level, the biological system studied –self-incompatibility- is an essential feature of plant mating systems, whose manipulation could lead to improvement of crop breeding procedures.

• What are the overall objectives?
We have taken a multidisciplinary approach combining theoretical and empirical population genetics, evolutionary genomics and ancestral protein resurrection. Our combination of various powerful approaches in a tractable model system has provided insight on diversification, a poorly understood but fundamental evolutionary process that is taking place at all levels of organization.
Workpackage 1.
• We published a comprehensive review on the evolution of self-incompatibility (Durand et al. Evolutionary Applications 2020)
• Evolutionary genomics. We developed a bioinformatic pipeline for the reliable identification of S-alleles (Genete et al. MBE 2020). This pipeline was used to determine the distribution of S-alleles in A. lyrata (Takou et al. MBE 2020) and A. thaliana (Tsuchimatsu et al. MBE 2017). We developed a sequence capture protocol to study polymorphism of the genomic region linked to the S-locus (Le Veve et al., in prep). In collaboration with the group of Dan Schoen, we showed that in the Leavenworthia genus some alleles of the LalSRK gene have been acquired through exaptation of a paralogous sequence (Chantah et al. New Phyt 2017). We assembled a draft A. halleri genome to study the dynamics of transposable elements (Legrand et al. Mobile DNA 2019). We revealed the rapid evolution of pollen gametic segregation distortion across the genome (Corbett-Detig et al. Evolution Letters 2019 and Carpentier et al. in prep). We collaborated on the study of the mating system determinants in the Oleaceae family (Saumitou-Laprade et al. 2017, Carré et al. 2021). Finally, we recently published a review on the challenges and opportunities offered by the study of special interest regions across the genome (Vekemans et al. Mol Ecol 2021)
• Theoretical population genetics. We found that population subdivision severely affects SI stability (Brom et al. Evolution 2019). We then asked whether the introduction of compensatory mutations could counteract the spread of non-functional SI mutants and lead to the creation of novel functional S-haplotypes (Stetsenko et al. in prep). On a more general level, we studied the conditions under which competing clones can invade, be maintained stably or replace each other (Billiard and Smadi Am. Nat. 2020). Finally, we studied the joint evolution of lifespan and self-fertilisation (Lesaffre and Billiard JEB 2020), the dynamics of deleterious mutations in perennials (Lesaffre and Billiard Am. Nat. 2021), and the evolution of somatic mutations in plants (Lesaffre BiorXiv 2020). We also published a commentary on a new model of adaptive introgression (Vekemans and Castric New Phyt 2021).
• We finally started a series of ambitious experimental approaches to test these predictions directly. Our analysis revealed a clearly asymmetrical diversification process by which one of the two S-alleles has retained the recognition specifity of their common ancestor, while the other one has functionally diverged (Chantreau et al. eLife 2019).

Workpackage 2.
• We produced an ambitious literature review on the evolution of genetic dominance (Billiard et al. Biological Reviews minor revisions)
• Evolutionary genomics. We analyzed polymorphism of microRNA and their mRNA target sites (M2 of Chloé Beaumont, PhD of Flavia Pavan). We sequenced a large number of BAC clones containing the S-locus from natural populations (PhD of Nicolas Burghgraeve). In the frame of the PhD project of Jörg Bachmann, we showed that self-compatibility in the Capsella orientalis genome is controlled by mutations to the S-locus itself, and that small RNA scould be responsible for the dominance of self-compatibility (Bachmann et al. New Phyt 2017). We showed that self-compatibility in the allotetraploid species Arabidopsis suecica could be a secondary side effect of the dominance of the non-fonctional S-allele it received from its A. thaliana progenitor (Novikova et al. MBE 2017). In collaboration with Tanja Slotte we showed that the dominance hierarchy between six S-alleles has remained unchanged between Capsella and Arabidopsis (Bachmann et al. in prep).
• Theoretical population genetics. Audrey Le Leve compared the fate of mutations controlling the dominance/recessivity interactions in different manners (Le Veve et al. a in prep). She also developed simulation models to investigate the dynamics of neutral and deleterious mutations linked to dominant vs. recessive S-alleles (Le Veve et al. b in prep).
• On the functional side, we determined the molecular alphabet of the silencing phenomenon (Burghgraeve et al. Genetics 2020). We have established the dominance hierarchy between 11 S-alleles, confirming that co-dominance is rare (Durand et al. in prep). Contrary to our prediction, our results suggest that the canonical RdDM regulatory pathway is not involved in this regulatory interaction, challenging the current view in the field (Batista et al. in prep).
Our ambition was to address consequential evolutionary questions on plant sexual reproduction by integrating approaches ranging from plant molecular biology (to functionally characterize the genetic elements involved) to mathematical modeling to predict the action of natural selection on the variants observed in natural populations. Thanks to the ERC grant, we have been able to successfully develop a series of methodologies that are either entirely new, or that had not been implemented in the field before :
- innovative bioinformatic developments to genotype the S-locus of large series of individuals from raw resequencing reads
- ancestral protein reconstruction to test
- genetic transformation of Arabidopsis thaliana with the individual components of the self-incompatibility machinery (SCR, SRK and small RNA precursors)
- genetic edition by CRISPR/Cas9 of the main components of the RNA directed DNA Methylation pathway
- genomic library construction, allowing us unprecedented flexibility in the genome resequencing experiments