WP1 Genetics & Informatics is the hub of N2CROP, connecting all activities by delivering existing plant lines for testing in other WPs, collecting and integrating the resulting data, identifying candidate genes for genetic manipulation, and generating new plant genotypes using both existing EMS populations available from Traitomic and CRISPR/Cas9 gene editing. A key resource in WP1 is a faba bean diversity panel, which will be characterised at the genomic, transcriptomic and proteomic levels. WP1 will use machine learning-based multi-omics data integration combined with genome-wide association analyses and other quantitative genetics approaches to identify genes, proteins, carbohydrates and other compounds that affect target traits.

Stig Uggerhøj Andersen, AU
Kristian Barrett, DTU

Rhizobia labelled with molecular barcodes will be screened to identify specific symbiont strains that are able to outcompete less efficient local strains and provide the best performing nodules with the target plant genotype. On the plant side, suppression of nitrogen fixation by available soil nitrogen is a challenge, because the legume shuts down nitrogen fixation by forced nodule senescence and has to establish new nodules to re-initiate nitrogen fixation. As-yet unpublished discoveries at Aarhus University have identified central regulators of the nodule senescence induction mechanism and shown that their loss of function allows nitrogen fixation to persist during fertiliser application. These groundbreaking discoveries will be used to finetune the legume nitrogen engine of mixed cropping systems using targeted mutagenesis in faba bean, lupin and pea guided by structural insight into protein function.

Kasper Røjkjær Andersen, AU
Marcela Mendoza-Suarez, AU
Dorte Bodin Dresbøll, KU

The two crops in a mixed cropping system compete for resources, and the effects of alterations to legume nitrogen fixation and seed carbohydrate and protein loading on competitiveness, total yield and nitrogen uptake of the partner crops are complex. N2CROP will deploy carefully designed field experiments coupled with crop modelling to predict effects of specific perturbations and guide the molecular genetic approaches. Field and controlled pot experiments will be carried out at SLU and KU to evaluate the growth, nitrogen economy and grain yield in intercropping systems and the corresponding sole crops. Sole faba bean crops and faba bean–wheat and faba bean–oat intercrops will be grown using different legume lines, including mutants. Nitrogen-use efficiency in the intercrops and sole crops, will be evaluated using growth and trait space analysis. The resulting data will be used to optimise modelling of intercropping systems. The effects of nitrogen fixation-enhancing mutations, different branching patterns, root characteristics on performance when grown as sole crops or cereal–legume intercrops will be predicted. The crop modelling will result in the prediction of the desired characteristics of nitrogen fixation regulation mutants, and of optimal intercrop fertilisation regimes, planting patterns and densities.

Martin Weih, SLU
Dorte Bodin Dresbøll, KU

Katharina Meurer, SLU
Stig Uggerhøj Andersen, AU

Most well-adapted faba bean cultivars have a protein content of ~29%. Screening a broader diversity panel, we found variation in protein content from 24% to 39%, indicating that it is possible to increase protein content up to 30% compared to current cultivars. WP5 and WP6 will discover and annotate carbohydrate active enzymes using novel functional prediction tools, quantify seed carbohydrate composition and protein composition and bioavailability for the faba bean diversity panel from WP1, benchmarking against other popular protein sources such as whey and soy. Likewise, the content of antinutritional factors will be analysed. Cell wall carbohydrate compositional analysis will be performed, protein digestibility and release of nutritionally available amino acids during digestion (PDCAAS) will be analysed using optimised INFOGEST protocols, and a cell-free model simulating microbial enzymatic degradation in the gut will be established. The degree of digestion will be determined and digestates will be analysed in in vitro assays and cellular models for antioxidative, antihypertensive, and antidiabetic activities. A subset of 20-40 lines will be analysed for their impact on an established cellular model of the developing intestine. Data will be supplied to WP1 for identification of genetic regulators, and mutants from WP1 will be tested to verify gene function hypotheses and deliver legumes with higher protein content and bioavailability.

Jesper Holck, DTU
Esben Skipper Sørensen, AU

WP7 Management handles scientific coordination, overall project financial and scientific reporting, organisation of partner meetings and workshops, and an international conference on legume innovation in agri-food systems. An international advisory board has been established to monitor progress and further extend the reach of networking activities.

Stig Uggerhøj Andersen, AU
Martin Weih, SLU
Jesper Holck, DTU
Esben Skipper Sørensen, AU
Taylor FitzGerald, AU