Given the complexity of the problems to tackle with, to ensure IMPRESA achieves its objectives only holistic, multidisciplinary approaches are expected to provide with effective solutions at the regional and trans-national levels. The overall methodology falls within research activities, both of more basic nature and of practically (breeding) oriented. The methodological workflow is envisaged as an integration of horizontal activities, using the same genetic stocks to collect information from many trials/analyses, and vertical activities, focusing on targeted genomic regions of DW and WWRs to understand the mechanisms determining the expression of tolerance to targeted stresses, make use of associated molecular markers (available in some cases, to be found in others), and eventually identify candidate genes/QTL. 

The “horizontal” approach is well exemplified by the field trials (see WP2), which will be carried out by all participant countries. Screening the same materials (see below) and traits across a range of Mediterranean environments, from more favourable ones (Italy) to those more deeply affected by multiple stresses (Algeria, Tunisia, Turkey), as well as to alternative agricultural practices, will not only improve our understanding of how all the individual traits are integrated by the growing plant to produce a particular yield in a particular environment, but also enable to identify the most stable stress- and yield-related traits and QTL across environments, or traits/QTL that are expressed in an environment-specific manner. Still cross-cutting (= horizontal) is considered the approach of subjecting a substantial sub-set of the genetic materials available to the project to a panel of physiological tests under controlled conditions, aiming to assess the response of critical plants organs for yield formation (reproductive organs, flag-leaves, roots) to major stress conditions, such as heat, drought, heat+drought, and salt.

The information from screenings in multiple natural environments and controlled stress conditions will represent the basis for one of the methodological approaches that will be pursued to exploit the identified positive traits, called “introgressiomics” (Prohens et al. 2017 Euphytica 213:158), consisting of an even mass-scale development of plant materials carrying introgressions of genome fragments from crop relatives into the genetic background of crop species (see WP5). This approach of “pyramiding” of genomic regions of interest is particularly beneficial for breeding interventions aiming at improving complex traits, such as response to abiotic stresses or yield-related parameters, or when multiple traits are targeted. The latter is expected to be the case for DW-WWR amphiploids (see below), containing the whole genome of a wild relative, adapted to a variety of stressors, hence likely possessing positive attributes towards multiple stresses. The introgressiomics approach is largely pursued resorting to chromosome engineering (CE) strategies, based on the use of DW mutants lacking the main wheat gene, i.e. Ph1, controlling meiotic pairing between homoeologous (similar, though not completely homologous) chromosomes of wheat and of the wild relative (reviewed in, e.g., Ceoloni and Jauhar 2006, Genetic Res., Chrom. Engin. and Crop Improv. CRC Press, pp. 27-59). DW-WWR recombination can occur when Ph1 is absent, giving rise to balanced products where segments of wheat chromosomes are replaced by corresponding ones of the wild homoeologues. Several of the DW-WWR amphiploids included in the IMPRESA set of materials have been already put in suitable Ph1-lacking backgrounds, or contain homologous genomes shared between the DW and WWR parents (no major obstacle to recombination). DW-WWR amphiploids will be characterized through cytogenomic analyses. Focusing on those that will show interesting traits based on physiological screenings, these will be firstly characterized at the whole genome level via Fluorescent Genomic In Situ Hybridization (F-GISH) techniques. Subsequently, complete or recombinant chromosomes whose presence will be associated with stress-tolerant phenotypes in backcross progeny to DW, will be identified via FISH with chromosome-specific probes and eventually isolated through flow-sorting techniques for sequencing and throughout molecular characterization (WP5). 

On others of the particular genetic stocks that the project will exploit will be subject to “vertical” analytical and harnessing approaches. These are the stable DW recombinant lines previously produced by P0 members through CE strategies, and incorporate single alien chromosome segments transferred from a given wild related species, notably Thinopyrum ponticum (or, in one case, from two species of the same Thinopyrum genus, but combined in a single segment). The alien gene content of such genotypes has been well characterized as for disease resistance, quality and yield-related traits (Gennaro et al. 2007 doi:10.1560/IJPS.55.3-4.267; ibid. 2009 doi:10.1007/s10142-009-0115-1; Kuzmanovic et al. 2014 J Exp Bot 65:509 ; ibid. 2016 Field Crops Res 186:86). Thus, already for these traits of wide efficacy and validity for breerders and consumers across environments, such DW recombinant lines could be used in the project activity aimed at developing novel breeding materials using suitable recipient DWs of the various partner countries. However, at least for some of them, evidence for a contribution to important traits expressed in stressed environments of additional genes/QTL present in the same alien segments, makes these genotypes a relevant focus material for multifaceted improvement of weakly performing cultivars of various countries. Moreover, for several recombinant types, near-isogenic recombinant lines (NIRLs) have been developed in a homogeneous genetic background, which makes them particularly suited for any differential analysis. The comparison, in fact, will be in this case largely limited to the presence vs. absence of the alien segments (NIRL pairs, with and without a given alien segment), and hence to restrict to the specific alien segment introgression any variation vs. the non-carrier (normal DW) line resulting from the comparative physiological (WP3) and “omics” (WP4) analyses. 

As to the latter ones, a top-down approach will be followed, starting from development of metabolomic profiles. In fact, metabolomics is not dependent on the availability of reference genomes; further, the “untargeted” method, as in the IMPRESA case, is comprehensive, high throughput and facilitates the discovery of novel biomarkers for the molecular phenotypes of plants in response to abiotic stresses (Arbona et al., 2013, Int J Mol Sci 14:4885). The expected huge amount of variable metabolites will be progressively down-scaled through focus on major pathways involved in the stress response, to corresponding proteins and ultimately candidate genes, analysing the data sets in silico by using available data bases and sequenced reference genomes of Triticeae species, e.g. rice, barley, maize, cultivated wheat (Triticum aestivum, T. durum) and wild wheat relatives (T. dicoccoides, T. urartu, T. monococcum, Aegilops tauschii, Brachypodium distachyon).