The project is divided into correlation, causation and implementation phases. At Month 36 we are finalizing the correlative phase consisting of field trials including 600 barley lines grown under contrasting drought regimes in Austria, Morocco and Lebanon. Data were collected in the growing season 2023 on: i) below- and aboveground phenotypic responses such as biomass, yield, and root architecture in the plots; ii) microbial characterization in each barley plot, in total 10,800 samples that have been DNA sequenced for bacterial and fungal profiling; iii) 50 barley lines have been whole genome-sequenced, while app. 400 lines have been genotyped by SNP analysis. This massive amount of data has been analysed with the aim to identify genomic regions that are important for microbiome assembly and for drought resilience in barley, and also to identify microbial taxa that are responsive to barley genotype and drought conditions. We have identified SNPs that affect plant performance/drought responses by GWAS and likewise SNPs that affect microbial composition of barley roots have been identified. Last, we have identified SNPs that associate with both plant performance and microorganisms. By aligning fully sequenced barley genomes with SNP information these can be mapped to specific barley genes - 14 putative genes have been identified that are avaiting validation.
The identified genes will be knocked out using molecular transformation technology. The identification of target genes has been delayed, but knock outs of other genes likely to be involved in drought-responses and/or root architecture, have been produced for testing microbiomes and drought responses. The production of new lines takes app. 6 months, so we expect lines with the disrupted genes that have been identified in this project to be ready summer 2026.
Moving into the Causation phase, we have performed two controlled pot experiments with selected barley lines. Lines were selected from the Diversity and Spring panels based on variation in biomass and microbial composition. and interesting phenotypic traits, i.e. contrasting rhizosheath formation, stable and high biomass production across contrasting field trials, and finally we include mutant lines produced by AU in WP7. Experimental protocols (drought treatments, sampling and sample processing etc.) as well as pipelines for the integrated analyses of multi-omics data have been implemented and refined. At this point, we have identified specific drought-responsive metabolites and multiple drought-responsive microbial taxa and microbiome functional genes.
Root phenotyping tools have been developed enabling monitoring of root architecture from seedling to maturity. BOKU has developed a novel root imaging tool, DDrC, which enables continuous imaging of root growth under controlled conditions to evaluate stress adaptation plasticity of different barley varieties during the sensitive seedling establishment phase. PSI has developed an efficient software that enables efficient root image analysis. Furthermore, PSI has set up a phenotyping pipeline to monitor plants under drought and used this system to analyse 10 barley lines from our panels to track root stress adaptation at high spatial resolution.We have initiated work in the Implementation phase, where crossings of interesting barley lines (based on microbiome composition, biomass under drought, rhizosheath formation and rooting depth) has been performed, resulting in 90 crosses from 12 elite lines and 24 donor parents. At the moment, KASP markers specific to introgressions are being developed.
