FACCE ERA-NET+ GrassLandscape (Project Leader: Jean-Paul Sampoux, INRA, France)

Our project aims to implement an innovative methodological frame to screen the natural diversity of a
grassland species in order to discover genetic variability involved in environmental adaptation, and more
specifically in climatic adaptation (Sampoux et al., 2013). We will consider the use of results delivered by this
approach to plan strategies to restore permanent grasslands degraded by climatic shifts and disruptions. Our
project will focus on perennial ryegrass (Lolium perenne L.), which is a major grass species naturally
distributed over the whole European continent as well as in Northern Africa and Near East (Humphreys et al.,
2010). This species is a major component of many natural grassland communities. It is a resource capture
strategy species (Martin et al., 2009) which is especially prevalent in grasslands grazed by cattle. It is also the
main grass species sown in Europe to create temporary meadows and it has therefore received extensive
breeding effort during the last four decades (Sampoux et al., 2011). Recent developments in a new area of
ecological sciences (landscape genomics) have paved the way to the discovery of genomic markers of
adaptive diversity from genome-wide genotyping data. They are based on the implementation of methods
correlating genomic polymorphisms and environmental variations at sites of origin of genotypes combined
with tests of signature of selection (Manel et al., 2010). We will implement this methodological frame to detect
genomic markers of climatic adaptation in the natural diversity of perennial ryegrass. We will use a genotyping
method based on massively parallel sequencing technology applied to a high number of populations obtained
from genebanks of plant breeding research institutes or collected in situ across Europe. We will furthermore
take advantage of know-how and facilities of plant breeding research institutes participating in the project to
phenotype these populations in fields and in controlled environment to record agronomic and ecophysiological
traits. These phenotypic data will be used to model associations between phenotypic variability
and genomic polymorphisms. Association models between genomic polymorphisms and environmental variations will be used to map
the spatial distribution of genomic markers linked to adaptive diversity in present climatic conditions and to
foresee possible shifts in the spatial range fitting these markers in the context of several climate change
scenarios based on the four Representative Concentration Pathways (RCP) of IPCC AR5 (Moss et al., 2010).
On the basis of these results, we will define allelic profiles of perennial ryegrass expected to provide climatic
adaptation at regional scale over Europe under the future climatic conditions foreseen by climate models. We
will consider combining climatic adaptation and value for services (forage production and climate mitigation)
by the recombination of alleles providing climatic adaptation and value for services. We will design several
genetic pools mixing different natural populations for breeding regionally adapted populations to restore
permanent grasslands degraded climatic disruptions. Breeding and releasing improved genetic material will
be out of the scope of the project but will be discussed with stakeholders at the end of the project during ad
hoc meetings for implementation in further collaborative projects.

Natural populations of perennial ryegrass have been extensively collected during the last three decades as
potential genetic resources for breeding programmes and have been maintained as seed lots (accessions) in
genebanks of plant breeding research institutes. Genebanks thus provide a straightforward access to a large
number of natural populations of perennial ryegrass. We will establish a "genebank set" by choosing 500 accessions
originating from sites that will cover the distributional range of perennial ryegrass. We will also set up a second
diversity test, by performing a new in situ sampling of 50 populations across the same territory that will maximise
environmental variations. Sampling will be performed by collecting tillers from 30 plants in each of the 50 populations.
Geographical coordinates of collection sites of populations will be used to extract climatic data from
climatic databases. Data for other environmental parameters possibly involved in the spatial
distribution of adaptive diversity will also be collected, along with field performance data (including NIRS measures of
forage traits). Plants will be genotyped using Restriction-Associated DNA (RAD) sequencing on an Illumina HiSeq2500
platform to obtain ~70,000 genome-wide SNP markers. Resequencing of several candidate genes putatively associated
with environmental adaptation (i.e. vernalisation) will also be conducted. Associations between genotype
and phenotype will be detected using developing models for changes in allele frequency within mixed swards.

Our project will be among the first ones to implement the landscape genomics approach with a so extended protocol (a large set of populations covering the whole area of primary expansion of a species and a genomewide genotyping providing several tens thousands of genomic markers). It should then release results that will be worthwhile to publish in high impact journals of Ecology such as Trends in Ecology and Evolution, Ecology Letters, Molecular Ecology and Ecography. Results about shifts with climate change in the spatial distribution of perennial ryegrass adaptive diversity could be published in journals like Global Change Biology, Climatic Change, Agricultural and Forest Meteorology, and Regional Environmental Change. The outcomes of our project in the domain of conservation and use of genetic resources and in plant breeding, notably the added value of the landscape genomics approach in these fields, could published in journals like Theoretical and Applied Genetics, Molecular Breeding and Field Crops Research.

Results in the scope of landscape genomics and molecular ecology could be communicated at international conferences such as the VIPCA conferences. Results regarding genetic resources and plant breeding could be communicated at conferences such as the Eucarpia conferences (especially those of the forage plants section and genetic resources section), the MBFT(molecular breeding of forage and turf) conferences and the European Grassland Federation conferences.

Communication towards stakeholders will be a point of focus in our project. It will be the target of task 4.3 in WP 4. WP 4 will have the objective to map the spatial range fitting genomic markers of adaptive diversity in present climate and in several climate change scenarios. WP 4 will also aim to design genetic pools mixing natural populations bearing different climatic adaptations. These genetic pools will have to be submitted to a number of selection cycles to recombine allelic diversity for climatic adaptation and value for services. Selection in these genetic pools is intended to deliver improved populations for restoring permanent grasslands with genetic material adapted to future regional contexts of Europe (see Figure 1 in part "Expected impact of the project"). Our project will include the design of these genetic pools. However, the practical setup of these pools will be out of the scope of the project. This will have to be undertaken, as well as further breeding, in next projects beyond the end of this FACCE-JPI project. Such tasks involving extensive plant breeding logistics cannot be carried by the sole academic partners of the project and will have to be implemented in close collaboration with other stakeholders, including plant breeders, technical advisers and farmers. Our communication towards stakeholders will not only have the objective of transferring our findings, but also to prepare a practical followup including the setting up of pools and breeding in collaboration with stakeholders.

We plan to implement our communication strategy towards stakeholders by the medium of professional societies. We will consider the grassland societies gathering professionals interested in grasslands (farmers, technical advisers, scientists, plant breeders). All these societies have their own regular conferences and web sites to disseminate information. The national grasslands societies that we can address are the British Grassland Society in the UK, the 'Arbeitsgemeinschaft Grünland und Futterbau' (AGGF), the 'Gesellschaft für Pflanzenzüchtung' (GPZ) and DLG - 'Fachzentrum Land- und Ernährungswirtschaft' in Germany and the 'Association Française pour la Production Fourragère' (AFPF) in France. National grassland societies from other countries could possibly be contacted. We will also communicate towards national plant breeder societies.