Towards the first goal (1), we re-sequenced using short reads the genomes of ~1,000 field-collected individuals of A. halleri, for most of which we had also quantified multiple relevant plant and environmental parameters in a field survey (Task 1.1). Based on SNP polymorphisms, we revealed details of population structure and demography, and we completed genome-wide association analyses employing various parameters measured in the field for 835 genotypes (DOI: 10.1111/nph.14219). We also conducted large-scale phenotyping experiments including hundreds of field-collected individuals under standardized environmental conditions in a growth chamber. Furthermore, we mapped QTL for metal hypertolerance and leaf ionomic traits in F2 populations of targeted crosses between phenotypically contrasting accessions of A. halleri upon plant cultivation on appropriately composed artificially metal-contaminated soils (Task 1.2). We identified putative causal loci and polymorphisms within QTL, and also within GWAS peaks, and we addressed and, in part, obtained experimental evidence for their biological roles for a subset of them. Our genetic linkage mapping identified QTL regions contributing to the hyperaccumulation of cadmium, a trait which we observed to be confined to only a subset of phylogenetic lineages of A. halleri. Based on another cross, the approach identified QTL regions underlying locally enhanced metal hypertolerance on an extremely highly metal-contaminated soil.
The generation of a number of haplotype-phased genome assemblies of A. halleri genotypes (for example,
https://phytozome-next.jgi.doe.gov/info/Ahalleri_v2_1_0(si apre in una nuova finestra)) which are central for our project enabled us to examine the gene content of the identified QTL and, as a first step, of an exemplary subset GWAS peaks. Thus, we identified candidate genes and polymorphisms, for which we subsequently gained additional evidence using a variety of approaches, including also functional experimental confirmation. These results allow us now to generalize on the genetic architecture of local adaptations in A. halleri and the types of polymorphisms and functional alterations of genes selected for. They also allow us to compare between our QTL mapping and the GWAS approach, allowing conclusions on the origin and evolutionary trajectories of the alleles conferring adaptation.
Towards the second goal (2), we have completed obtained transcriptome data for root and shoot tissues of a large number of genetically diverse A. halleri individuals following cultivation on two contrasting soil types in a growth chamber for RNA extraction and sequencing-based transcriptomics (Task 2.1). For a set of four phenotypically contrasting exemplary accessions of A. halleri and A. thaliana (Col-0), time course experiments of the depletion and excess of single metal ions in hydroponics are completed with all replicates (Task 2.2). Transcriptome data are available any analysed for the subset of excess metal treatments. Towards goal (3) we were able to complete a series of experimental evolution experiments and are preparing for the analysis of plants at the genome level.
Results addressing goal (1) were presented at a number of conferences, and two major manuscripts are at an advanced stage of preparation for publication. Results towards goal (2) were presented at several workshops, and we have begun to work on a manuscript. The results towards goal (3) will require some further work. We are also preparing the genome assemblies generated in this project for their public release. Manuscripts containing small subsets of results for all goals have been published.
