Comparative population genomics of red clover domestication and improvement

We propose to use the largely undomesticated red clover forage crop as a model for unravelling a key domestication trait. Forage legumes have superior feeding value for ruminant animals, and their nitrogen fixing capability enables them to provide useful ecosystem services in terms of improvement of soil fertility. Despite these properties their use in livestock agriculture declined particularly in Europe in the 70's and 80's, chiefly due to the availability of cheap chemically produced nitrogen fertilizer. The drive towards more sustainable agriculture, particularly less use of fertilizer manufactured from fossil fuels has halted the decline, and there is increasing interest in these legume crops, particularly in mixtures with forage grasses. There is thus an urgent need to accelerate their genetic improvement, which has stalled in later years due to lack of investment. This proposal aims to use recently developed genomics resources for red clover, second only to alfalfa in importance in temperate agriculture, to assess the genetic and phenotypic diversity of a European-wide collection of germplasm. One of the most fundamental requirements for genetic improvement programmes is to have access to genetic variation within your germplasm. There are suggestions that most recent European breeding populations have a relatively narrow base. With a very recent history of breeding, the largely undomesticated red clover crop is an ideal candidate to provide a comprehensive assessment of the role of domestication in changing the genome landscape during a crop improvement programme. In other words we will aim to characterise the genomic impact of domestication in a crop improvement programme by using red clover as a model.
We will use a collection of populations from a range of habitats from throughout Europe together with elite breeding material. We will use this diversity panel to assess the genome-wide nucleotide diversity and use this information to tell us which regions of the genome have been subject to selective pressures either as a result of breeding or environmental adaptation. The focus will be on a key domestication trait, namely prostrate versus erect growth habit, which has a profound effect on grazing tolerance and persistency in forage crops. Plants with more prostrate growth habits are likely to be more tolerant to grazing and be more persistent. On the other hand, there is a yield penalty associated with prostrateness. Unravelling the genetic architecture is thus of major importance for genetic improvement, and will also give us novel insight into this fundamental trait in plants.
We will use two types of plant material for this: A diversity panel consisting of ecotypes and natural populations with varying degrees of prostrate growth habit, and compare with elite breeding populations, all of which are erect. Secondly, we will generate populations segregating for this trait by crossing an erect female parent from elite material with five pollen donors taken from the prostrate natural populations. Phenotypic analysis of agronomic and growth traits in these populations will be accompanied by chemical analysis of various forage quality traits, and by obtaining genome-wide SNP polymorphism data. This will be achieved by restriction associated DNA (RAD) marker analysis in the mapping populations, as well as the diversity panel. In combination with the improved genome sequence assembly, this will enable us to identify and map genomic regions under selection, and allow identification of some of the genes governing this trait. This will provide novel insight into the architecture of domestication traits. The partnership with Germinal Holdings Ltd gives us a pipeline into the breeding programme, which will ensure that the genomic data and knowledge we obtain will benefit genetic improvement of red clover.

The aim of this project is to characterise the genetic diversity in natural and breeding populations to identify genome wide changes during breeding of red clover. We wish to investigate to what extent artificial selection has affected the genome including genic and non-genic regions, and whether this has resulted in a reduction in diversity and increase in rare alleles. We propose the following programme to test this. Firstly, we will use RAD marker polymorphisms from an existing mapping family to improve alignment of the red clover genome assembly to a genetic map. Secondly, we will use a 600 genotype strong panel of ecotypes with mostly prostrate growth habit together with erect elite breeding material to analyse the genetic diversity by RAD marker sequencing. Phenotypic, chemical analyses and ecogeographical information of the diversity panel will allow us to obtain information on how haplotype structure correlates with phenotype and environmental gradients likely to impact on environmental adaptations. Nucleotide diversity and locus by locus genetic differentiation will reveal genomic regions under selection. We will also generate mapping families segregating for growth habit, thus enabling us to associate and map more accurately the target traits relating to a prostrate and erect phenotype. The goal is to identify the genes responsible for most of the genetic variation in this key domestication trait. This project will generate information on the genetic basis of a fundamental trait, provide insight into selection during recent domestication and inform the forage legume breeding programme. We will also re-sequence the five pollen donors in the new mapping family. This, the RAD marker data and the reference assembly will give us valuable information about genome-wide linkage disequilibrium, levels of heterozygosity, SNP density and patterns of polymorphisms in coding and non-coding sequence.

The delivery of the proposed objectives is focused on current needs for which suitable tools and skilled researchers are lacking or not fully developed. The objectives will also directly contribute solutions in areas of research relevant to the BBSRC's scientific priorities in food security and integrate established methods in genetics with emerging multidisciplinary research areas such as genomics and bioinformatics.
This programme will generate new opportunities for collaborative work between TGAC and IBERS and will also extend to other R&D groups in industry and academic institutions. We will communicate our results of fundamental aspects of domestication traits in forage crops through peer reviewed publications, and at national and international conferences. This work will significantly improve the draft red clover genome, which will be one of the first for a temperate forage crop. It will thus have general interest beyond the immediate circle of colleagues interested in red clover. We will disseminate the data using TGAC computing resources and by depositing the raw sequences and assemblies in long-term repositories established at the EMBL-EBI. One of the main stakeholders is Germinal Holdings Ltd who funds a significant part of IBERS breeding programme. This work is aligned closely with the initial stages of the new red clover breeding programme. The generation of large numbers of SNP marker polymorphisms will bring with it the potential to use genomics-based prediction of selection candidates. Accelerated breeding cycles lead to a faster route from breeding material to new varieties. The current programme of genetic improvement in red clover has the potential to lead to significant increases in seed sales, of which Germinal Holdings have 18% currently in the UK. New varieties with better persistence and disease resistance also open up significant opportunities for export to the European market, as well as North America and New Zealand.

The proposed work will directly impact the local community with the generation of new jobs and potential funding opportunities. The collaboration between IBERS and TGAC has strengthened the position of these Institutes in hosting specific expertise in advanced breeding for forage legumes. This will create new opportunities around the development of applications in genomics and bioinformatics, which will translate into job opportunities. It is widely recognised that the shortage of expertise and skills in biomathematics and informatics in the UK and across the world is a major risk for future development in life sciences, in this context this proposal will attract talented staff to work with IBERS and TGAC. Finally, the ecotype collection we use here consists partly of natural populations that were collected from locations in Eastern Europe in the period from the early 1990's to the early 2000's. Many of these habitats are now under severe threat, which means that some of the accessions we work with may no longer exist naturally. Our work thus represents the best way of utilising this unique resource, and preserving the diversity.

This collaboration will contribute to reinforce the UK's leadership in translating the development of genetic and genomic resources from fundamental science to applications with a potential impact on the local and national economy. The development of a more sustainable agriculture is a key aspect of the UK strategy and this is aligned with the objectives in the recently published Agri-tech Strategy. A consequence of the implementation of this proposal is to position IBERS and TGAC as international leaders in biotechnology specifically in the area of forage legumes. This will deliver impact to a broad range of stakeholders emphasising the key role that the Institutes will play in enabling researchers to develop cutting-edge science in the coming years, and ensure that the genomics resources will be translated to research and breeding programmes.