Comparative population genomics of red clover domestication and improvement

Lead Research Organisation: Aberystwyth University
Department Name: IBERS

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description One of the objectives was to analyse the diversity of European and Asian red clover germplasm in terms of genotype and phenotype. We have obtained and analysed genotypic data (12K SNP markers) using genotyping by sequencing technology, and we have phenotypic data (phenology and morphology) from one years worth of field trials at IBERS. The results were presented at the Plant and Animal genome XXV conference in January 2017, and a peer-reviewed publication is in preparation. Through genome wide association analysis we have identified a number of potential candidate genes associated with some of the traits we have measured. Phenotypic data will be recorded again in 2017 to allow a robust association analysis.
Another objective was to generate a nested pseudoNAM population in which an elite motherplant was crossed separately with 5 morphologically different pollen donors in order to obtain populations segregating for architecture. This has been achieved, and 100 progeny from each of the five families have been planted in spaced plant trials in the field in a RCBD. Phenotypic data (phenology and morphology) have been recorded for one year, and genotypic data (GBS) are being generated. The five parents have also been resequenced, and will serve as reference for SNP tags for the mapping families.
Thirdly, we have generated a much more dense genetic map of another mapping family through GBS data which will allow us to update the reference genome sequence of red clover. A new version (v3.0) of the genome assembly has now been generated and will be publically available in the near future.
Exploitation Route The findings and promising lines of red clover are now used as a basis for new, more grazing tolerant varieties in IBERS Forage Plant Breeding programme. One of the populations used in the generation of mapping families is used in the breeding programme as a basis for this selection and breeding goal.
Sectors Agriculture, Food and Drink,Education,Environment

URL https://legumeinfo.org/genomes/gbrowse/Tp2.0
 
Description The project has resulted in in-depth genomic analysis of European and Asian red clover germplasm. This has been accompanied by some phenotypic data. This in turn led to the selection of promising creeping or prostrate red clover ecotypes, which have been used in further genetic analysis using mapping populations. The results have been presented at various international conferences, and will be published in peer-reviewed journals shortly. Furthermore, we now have a large collection of SNP marker data with which to analyse and assist in our breeding programme. One of the accessions has now been taken forward in the breeding programme for grazing trials, and is due to be put forward in national list trials for variety acceptance.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Education,Environment
Impact Types Economic

 
Description AberForward graduate placement scheme
Amount £1,500 (GBP)
Organisation Aberystwyth University 
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 02/2017
 
Description KESS II
Amount £52,000 (GBP)
Organisation European Social Fund (Welsh Government/ EU) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description SMARTExpertise
Amount £500,000 (GBP)
Funding ID SMARTExpertise - 2017/COL/008 
Organisation Government of Wales 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Sustainable Food Security - Resilient and resource-efficient value chains (H2020-SFS-2016-2017)
Amount € 5,000,000 (EUR)
Funding ID H2020-SFS-2016-2 727312 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 09/2017 
End 08/2021
 
Title DNA sequence of red clover genome 
Description DNA sequence data from red clover 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact The paper with the data has been accessed and viewed many times: •in the 98th percentile (ranked 2,876th) of the 177,703 tracked articles of a similar age in all journals •in the 97th percentile (ranked 59th) of the 2,199 tracked articles of a similar age in Scientific Reports The altimetric score is 107. The paper has been tweeted by 23 Blogged by 2 Picked up by 11 news outlets It has been viewed 1293 times (07/03/16) 
URL http://dx.doi.org/10.5281/zenodo.17232
 
Description Colombia and Kenya Partnering Award: Skills sharing for genomic approaches to forage improvement 
Organisation CGIAR
Department International Center for Tropical Agriculture
Country Colombia 
Sector Charity/Non Profit 
PI Contribution Participation in workshop on genomic selection assisted breeding of tropical forage crops
Collaborator Contribution Breeder participation and visit for training purposes
Impact Visit of plant breeder from CIAT to TGAC in the summer of 2016. Forages for Africa programme also initiated with participation from IBERS.
Start Year 2014
 
Description Colombia and Kenya Partnering Award: Skills sharing for genomic approaches to forage improvement 
Organisation Earlham Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Participation in workshop on genomic selection assisted breeding of tropical forage crops
Collaborator Contribution Breeder participation and visit for training purposes
Impact Visit of plant breeder from CIAT to TGAC in the summer of 2016. Forages for Africa programme also initiated with participation from IBERS.
Start Year 2014
 
Description Colombia and Kenya Partnering Award: Skills sharing for genomic approaches to forage improvement 
Organisation International Livestock Research Institute (ILRI)
Country Kenya 
Sector Charity/Non Profit 
PI Contribution Participation in workshop on genomic selection assisted breeding of tropical forage crops
Collaborator Contribution Breeder participation and visit for training purposes
Impact Visit of plant breeder from CIAT to TGAC in the summer of 2016. Forages for Africa programme also initiated with participation from IBERS.
Start Year 2014
 
Description Legume Steering Group 
Organisation EMBL European Bioinformatics Institute (EMBL - EBI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Member of the Legume Steering Group led by Prof Christine Foyer, University of Leeds. The purpose is to reinvigorate excellent legume basic research leading to BBSRC and other proposals; create a unified legume community; work with DEFRA to inform government on legume-related issues; attract new researchers to the legume community; engage with industry and policy makers in research, agriculture and health, organize a yearly scientific meeting. IBERS forage and pulse plant breeding team is keen to contribute to achieving these goals, and have participated in the latest steering group meeting held in London on February 20th.
Collaborator Contribution The steering group was formed recently. There are plans to hold a meeting at NIAB later in the year. Collaborations between IBERS and some of the members of the steering group has led to successful applications for BBSRC funding.
Impact The PeaGen BBSRC LINK project between IBERS, JIC and other partners was not formed as a result of the legume steering group, but can be said to be an example of what can be achieved, and has encouraged the forming the steering group, and shaped its membership.
Start Year 2017
 
Description Legume Steering Group 
Organisation James Hutton Institute
Country United Kingdom 
Sector Public 
PI Contribution Member of the Legume Steering Group led by Prof Christine Foyer, University of Leeds. The purpose is to reinvigorate excellent legume basic research leading to BBSRC and other proposals; create a unified legume community; work with DEFRA to inform government on legume-related issues; attract new researchers to the legume community; engage with industry and policy makers in research, agriculture and health, organize a yearly scientific meeting. IBERS forage and pulse plant breeding team is keen to contribute to achieving these goals, and have participated in the latest steering group meeting held in London on February 20th.
Collaborator Contribution The steering group was formed recently. There are plans to hold a meeting at NIAB later in the year. Collaborations between IBERS and some of the members of the steering group has led to successful applications for BBSRC funding.
Impact The PeaGen BBSRC LINK project between IBERS, JIC and other partners was not formed as a result of the legume steering group, but can be said to be an example of what can be achieved, and has encouraged the forming the steering group, and shaped its membership.
Start Year 2017
 
Description Legume Steering Group 
Organisation John Innes Centre
Department Department of Crop Genetics
Country United Kingdom 
Sector Private 
PI Contribution Member of the Legume Steering Group led by Prof Christine Foyer, University of Leeds. The purpose is to reinvigorate excellent legume basic research leading to BBSRC and other proposals; create a unified legume community; work with DEFRA to inform government on legume-related issues; attract new researchers to the legume community; engage with industry and policy makers in research, agriculture and health, organize a yearly scientific meeting. IBERS forage and pulse plant breeding team is keen to contribute to achieving these goals, and have participated in the latest steering group meeting held in London on February 20th.
Collaborator Contribution The steering group was formed recently. There are plans to hold a meeting at NIAB later in the year. Collaborations between IBERS and some of the members of the steering group has led to successful applications for BBSRC funding.
Impact The PeaGen BBSRC LINK project between IBERS, JIC and other partners was not formed as a result of the legume steering group, but can be said to be an example of what can be achieved, and has encouraged the forming the steering group, and shaped its membership.
Start Year 2017
 
Description Legume Steering Group 
Organisation National Institute of Agronomy and Botany (NIAB)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Member of the Legume Steering Group led by Prof Christine Foyer, University of Leeds. The purpose is to reinvigorate excellent legume basic research leading to BBSRC and other proposals; create a unified legume community; work with DEFRA to inform government on legume-related issues; attract new researchers to the legume community; engage with industry and policy makers in research, agriculture and health, organize a yearly scientific meeting. IBERS forage and pulse plant breeding team is keen to contribute to achieving these goals, and have participated in the latest steering group meeting held in London on February 20th.
Collaborator Contribution The steering group was formed recently. There are plans to hold a meeting at NIAB later in the year. Collaborations between IBERS and some of the members of the steering group has led to successful applications for BBSRC funding.
Impact The PeaGen BBSRC LINK project between IBERS, JIC and other partners was not formed as a result of the legume steering group, but can be said to be an example of what can be achieved, and has encouraged the forming the steering group, and shaped its membership.
Start Year 2017
 
Description Legume Steering Group 
Organisation University of Leeds
Department School of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Member of the Legume Steering Group led by Prof Christine Foyer, University of Leeds. The purpose is to reinvigorate excellent legume basic research leading to BBSRC and other proposals; create a unified legume community; work with DEFRA to inform government on legume-related issues; attract new researchers to the legume community; engage with industry and policy makers in research, agriculture and health, organize a yearly scientific meeting. IBERS forage and pulse plant breeding team is keen to contribute to achieving these goals, and have participated in the latest steering group meeting held in London on February 20th.
Collaborator Contribution The steering group was formed recently. There are plans to hold a meeting at NIAB later in the year. Collaborations between IBERS and some of the members of the steering group has led to successful applications for BBSRC funding.
Impact The PeaGen BBSRC LINK project between IBERS, JIC and other partners was not formed as a result of the legume steering group, but can be said to be an example of what can be achieved, and has encouraged the forming the steering group, and shaped its membership.
Start Year 2017
 
Description Legume Steering Group 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution Member of the Legume Steering Group led by Prof Christine Foyer, University of Leeds. The purpose is to reinvigorate excellent legume basic research leading to BBSRC and other proposals; create a unified legume community; work with DEFRA to inform government on legume-related issues; attract new researchers to the legume community; engage with industry and policy makers in research, agriculture and health, organize a yearly scientific meeting. IBERS forage and pulse plant breeding team is keen to contribute to achieving these goals, and have participated in the latest steering group meeting held in London on February 20th.
Collaborator Contribution The steering group was formed recently. There are plans to hold a meeting at NIAB later in the year. Collaborations between IBERS and some of the members of the steering group has led to successful applications for BBSRC funding.
Impact The PeaGen BBSRC LINK project between IBERS, JIC and other partners was not formed as a result of the legume steering group, but can be said to be an example of what can be achieved, and has encouraged the forming the steering group, and shaped its membership.
Start Year 2017
 
Description 12th British Grassland Society Conference, Aberystwyth, United Kingdom, September, 2015. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation of poster entitled "Comparative population genomics of red clover domestication and improvement" with results of research.
Year(s) Of Engagement Activity 2015
 
Description Bioinformatics for Breeding 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I was invited as an expert to teach at a three day hands-on course entitled Bioinformatics for Breeding held at the Earlham Institute Feb 21-24, 2017. It was primarily aimed at PhD students post doctoral researchers or early career scientists engaged in genetic mapping, QTL and association analyses, molecular marker identification and genomic selection.
Year(s) Of Engagement Activity 2017
 
Description Bioinformatics for Breeding 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Participated as a teacher on the Bioinformatics for Breeding course held at the Earlham Institute, Norwich over three days in February 2017. About 30 researchers with an active interest in how to use bioinformatics in breeding-related research such as next generation sequence data for genetic mapping, GWAS and genomic selection.
Year(s) Of Engagement Activity 2017
 
Description David Lloyd gave a talk to farmers group from Mole Valley Farmers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact David Lloyd, IBERS legume breeder gave a talk to a farming group from Mole Valley farmers about IBERS clover breeding activities
Year(s) Of Engagement Activity 2015
 
Description International Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of plenary talk at the EUCARPIA Joint meeting of Fodder Crops and Amenity Grasses Section and EUCARPIAs Protein Crops Working Group, September 11-14, Vilnius, Lithuania.
Year(s) Of Engagement Activity 2017
URL https://www.eucarpia2017.lt/
 
Description Lecture to MSc students, Aberystwyth University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Lecture by David Lloyd to MSc students BRM 1420 MSc Food Security
Year(s) Of Engagement Activity 2016
 
Description Presentation in workshop at Joint meeting of Fodder Crops and Amenity Grasses Section and EUCARPIAs Protein Crops Working Group, September 11-14, Vilnius, Lithuania 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Jones, C., Lloyd, D., Hegarty, M., De Vega, J., Ayling, S., Powell, W., Skøt, L. (2017). Population structure of red clover ecotypes collected from Europe and Asia. Proceedings of EUCARPIA Joint meeting of Fodder Crops and Amenity Grasses Section and EUCARPIAs Protein Crops Working Group, September 11-14, Vilnius, Lithuania (in press)
Year(s) Of Engagement Activity 2017
URL https://www.eucarpia2017.lt/
 
Description Talk to Irish dairy farming group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact David Lloyd gave a talk to Irish dairy farming group about the forage legume breeding activities at IBERS.
Year(s) Of Engagement Activity 2015
 
Description Training course at IBERS for 50 advisors 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Training for advisors relating to forage crop breeding and grassland management
Year(s) Of Engagement Activity 2015,2016
 
Description Training course sponsored by Germinal Holdings for seed merchants 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact David Lloyd, IBERS legume breeder gave a presentation at a training course sponsored by Germinal Holdings for seed merchants
Year(s) Of Engagement Activity 2016