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Periodic Report Summary 3 - WATBIO (Development of improved perennial non-food biomass and bioproduct crops for water stressed environments)

Project Context and Objectives:
The overall objective of WATBIO is to enable accelerated breeding for drought tolerance in three novel non-food crops for Europe. Our route to achieve this is to use an innovative approach to harness the power of next generation sequencing technologies to identify both single gene-trait associations for drought tolerance alongside the capture of natural genetic variation and allelic diversity, identify potentials for marker assisted breeding. We work together as a twenty three partner multi-disciplinary consortium where the inclusion of 7 SMEs that span the whole chain from fundamental science and molecular sequencing, to traditional and molecular plant breeding, biotechnology, plant propagation, commercial sales of plant material and an expert in dissemination and delivery, will ensure that the project achieves the overall aim of rapid deployment of new, improved plant material for non-food use suited to water stressed environments of Europe. We wish to develop more efficient breeding and genetic optimisation of these crops on unproductive land not utilised for food.
The project objectives of WATBIO are:
1. Fundamental understanding of drought signalling. To provide a better understanding of the fundamental mechanisms determining drought tolerance in Populus, Miscanthus and Arundo for the control of growth and transpiration efficiency in response to water stress (WP1, WP7)
2. A database of water-stress traits. To provide a database of ‘water stress response’ information, from laboratory to phenotyping platform to field for three novel biomass crops and quantifying these responses for a wide range of new germplasm developed in the project (WP2, WP7)
3. Innovative molecular breeding/genetic optimisation. To deploy next generation sequencing data, in 3 different, innovative approaches (RNASeq, GWAS and genetical genomics) for genetic optimisation and supply of new material in breeding programmes of partners within the consortium (WP3, WP4)
4. Innovative genetic optimisation using GM. To deploy GM technologies to test importance of single genes for drought tolerance, (WP3,WP5)
5. Environmental assessments of crops. To assess field performance of the new crop plants, in a range of environments, on marginal soils, spanning the European climatic envelope, to determine selection of superior genotypes with biomass production and biomass quality maintained (WP6)
6. To train the next generation of multidisciplinary non-food crop bio scientists through workshops, seminars and science exchanges and to ensure spreading of excellence between academics and SMEs (WP8)
7. To disseminate the project through 6 stakeholder workshops and additional ‘Local Fora’ meetings, both with appropriate literature and hand outs with all partners involved. To accelerate dissemination through the 23 partner critical mass developed in the project, using industrial partners for spreading excellence activities (WP9/10)
8. Interaction with SWEETFUEL, DROPS, OPTIMA, OPTIMISK, and a number of other EU projects. Achieve interaction, communication and cross-fertilization of ideas with current projects on sweet sorghum, maize, perennial grasses, Miscanthus and others, including organising a joint workshop (WP9)

Project Results:
Overall progress within WATBIO remains very good and all major objectives have been met with the project remaining largely on-track. There are however, some impacts of unforeseeable events largely related to unseasonable weather that have delayed activities but this should have no major effect on overall delivery of the project. To date, we have achieved a very large amount of work, with much engagement across the whole consortium as summarised in the periodic report, with an emphasis now on developing outputs and impact. Over twenty scientific peer-reviewed papers are already published, with many more planned and more than 90 presentations of WATBIO research have been made. To this end, in M59 a significant international conference is being organised in Oxford, UK to report the latest finding in bioenergy crop research bringing together specialists in approaches that range from genome editing to genomic selection and to consider all of the tools deployed in WATBIO for application to our three novel non-food crops. A Miscanthus safari was held in year 4 – a field trip that demonstrated new material of Miscanthus and on-going trials and a similar Populus safari in planned in Italy for 2017. The consortium has come together in an effective way and is working as a real partnership, achieved through the regular executive committee telecom meetings that take place each quarter where each WP leader reports major achievements and any deviations from plan, and so issues are dealt with effectively. There is a good working relationship between IT and SOTON to ensure effective project management and coordination.
For the whole project, a set of ‘core’ common genotypes for each species has been identified and throughout WATBIO, all partners are committed to working on this common material for the elucidation of fundamental insights into drought tolerance. In addition to this, a set of ‘extensive genotypes’ has been identified in all three species, several thousand for Populus, several hundred for Miscanthus and tens for Arundo on which to test hypothesis generated from our core material. In this way we hope to fulfil a major objective of WATBIO, to understand the genetic basis of drought tolerance and to accelerate breeding in this important area. In WP1, a large dataset of RNA-Seq data for transcriptomic responses of Populus, Miscanthus and Arundo has now been developed with data for all three species either submitted or accepted for publication. Extensive and highly controlled experimental work has been completed at INRA, IBERS-AU and CNR. In WP2, we have been working within two phenotyping platforms available in the UK and an extensive range of Miscanthus and Populus genotypes have been tested at AU-IBERS and LANCS respectively. In WP3 we have established a good working pipeline to ensure all requests for next generation sequencing are dealt with in a timely way by the Sequencing Request Group (SRG). More than 1500 samples have now been sequenced for RNA-Seq and a novel technology deployed in Populus for Genotyping by Sequencing (GBS) in collaboration with the company NuGen. In WP4, over 12,000 Populus trees have been planted to a strict experimental design informed by WU and these trees, representing over 1000 unique genotypes have been subjected to a controlled drought stress, during a second year, since in the first year of this experiment rainfall was higher than average. Even so, in the unique mapping population of black poplar UNITUS have collected data on drought tolerance and we are now planning the eQTL approaches in both Populus and Miscanthus. In Populus the GBS technique has been deployed to identify several new gene-trait associations including genes linked to drought tolerance. Similarly a novel technique for mutagenesis has been developed to generate 1000 new Arundo genotypes that have been assessed in the field and demonstrated in a ‘field day’ to industry. UNIBO and GeneticLab co-own this highly novel material and continue to work together on the the commercialisation of the material generated by the project. In WP5, underpinning work to develop transgenic lines of Populus and Miscanthus has been completed, utilising candidate genes identified from the literature, from preliminary GWAS and from the ‘Core’ experiments of WP1. A two way agreement is now being developed to protect some of these findings and more than 30 new GM Populus lines are now being distributed to partners. Whilst in WP6, several unique field experiments on all three species are now completed with data analysis in progress, following earlier delays of this task. A socio-economic analysis of the potential of these new non-food crops has been established. In WP7, two workshops have been completed on data analysis and techniques for RNA-Seq data analysis, GWAS, eQTL and network analysis and modelling that brings together phenotypic and gene expression data is now underway. In WP8, five training workshops have been completed. In WP9 the Communication Plan for the project has been written and the website and twitter feed are regularly updated. In 2016 several short films were made of the research activity of WATBIO and these are now tweeted and on YouTube, where WATBIO has a presence. A professional video is planned for 2017 to describe the project. Effective management of the project has been achieved through WP10.

Potential Impact:
We will develop the potential of three novel biomass crops, currently only planted over a few thousand hectares of land (Populus and Miscanthus) or hardly at all (Arundo) but for which considerable potential exists in marginal, drought prone soils. In workpackage 2, we are taking the very best available Phenomics Platforms to enable WATBIO to extend the analysis of workpackage 1, 3 and 7, with key traits identified, to develop strategies to measure these traits in a wider set of germplasm, again to improve breeding efficiency. The Lemna-Tec and other phenotyping platforms are only just emerging with very fast progress in the use of non-destructive remote analysis. The partnership has used a new development at AU-IBERS and a smaller facility at ULANC to make pioneering measurements of traits in our three species of interest. No such phenotyping is available for these crops. Our over-arching hypothesis in workpackage 1, is that although drought tolerance is a complex trait, difficult to elucidate, significance progress in recent years suggests that enough genetic diversity will exist in these species, to identify individuals (genotypes) that are able to maintain biomass productivity, biomass quality and water use efficiency, in droughted conditions and the plant attributes most likely to underpin these traits. All of these functional data will be utilised in workpackage 5 where the aim is to investigate key candidate genes in a standard genetic background using transgenic approaches. RNAi and Over Expression technologies in Populus will enable the project to assess rapidly the efficacy of particular genes in determining drought tolerance in Populus.
STT and DMB will use their expertise in the protection of IP and breeding rights to protect such plants and make them widely available for commercialisation through appropriate routes. We are fortunate that for two species, this direct technology from reverse genetics is available within the consortium and can also provide us with proof of concept for particular candidate genes. That proof of concept can be used within breeding programmes, to improve breeding efficiency, by searching for genes of interest from forward genetic approaches and the development of molecular markers, as described in workpackage 4. For our third species, Arundo, WATBIO will take an approach to generate increased genetic variation in this unimproved crop by completing a mutagenisation programme. Mutagensiation is used routinely in the majority of crop plants to generate such variation, but has never been applied to Arundo. Here in workpackage 4 in Populus, where the reference genome is available and where Taylor and Morgante in previous work have sequenced more than fifty genotypes, discovered 1 million SNPs and developed a genotyping SNP chip enriched in two genomic regions identified from QTL that are known to be associated with the water use efficiency trait of 13C, in WATBIO we will undertake a complete genome wide association study (GWAS) to link drought tolerance traits to underlying alleles. In Miscanthus and Populus a genetical genomics approach will be taken to identify cis- and trans-eQTL associated with drought tolerance. Given the importance of environment in determining the expression of drought tolerance traits, workpackage 6, where Dodd has extensive expertise of such experimentation and will develop a network of field sites that span the climatic envelope of Europe to test G xE interaction in the proposed starting and new material emerging from the project. This will provide a powerful dataset to be further interrogated by the model development in workpackage 7. The data of WATBIO are only of value if they have long-term Impact. This will be achieved in workpackages 8 and 9 where we will focus a significant resource of training and dissemination. Workpackage 9 is focused on securing the long-term economic, environmental and scientific impact.

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