Developing RAD markers as a resource for plant breeding

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

In order to improve the ability of plant breeding programmes to deliver the agricultural increases mandated by a growing population and changing climate, new techniques must be developed for rapid discovery and genotyping of genetic markers. The RAD (Restriction-site Associated DNA) sequencing (RADSeq) technique developed by Professor Eric Johnson of the University of Oregon, generates tens of thousands of genetic 'tags' from genomic DNA. Due to the capabilities of modern DNA sequencers, it is possible to sequence each of these tags many times and thus reliably spot genetic differences between two individuals. The capacity of second-generation sequencers is such that tags from multiple individuals can be pooled within a single sequencing run whilst still maintaining a high enough coverage of each tag to identify genetic differences. Tags from each individual can be identified by adding a unique 'molecular identifier' to the DNA prior to sequencing. By carefully selecting the right number of tags to be generated, it becomes possible to screen enough individuals within a single run to cover an entire genetic mapping population. RAD sequencing therefore combines the discovery, genotyping and mapping of genetic markers into a single step. Furthermore, if the phenotype of the samples is known, the data can be used to identify markers which segregate along with the phenotype, assisting in gene mapping and potentially gene identification. To date, RADSeq has primarily been used in animal or microbial systems. We propose to apply the RADSeq technique to a model cereal species, Lolium perenne (perennial ryegrass), in order to determine the applicability of this technique to improving plant breeding efforts. As a test case, we will use an existing mapping population designed to identify the two genetic loci controlling a self-incompatibility system in Lolium (ryegrass). We will perform RADSeq in the parents of this population at high coverage using two tag densities. We will then screen pooled mapping population progeny from each of four segregating genotypes (two per locus) in order to identify RADSeq markers which appear unique to each genotype. Finally, we will use the RADSeq marker information to construct a genetic map for this population and confirm the bioinformatic identification of a small subset of genetic markers using conventional genotyping. The proposed work will enable us to determine how well the RADSeq technique performs as a method for rapid marker discovery and genotyping in crops, using one of the most difficult examples - a highly heterozygous, outbreeding species. If RADSeq performs well under these conditions, it should easily be applicable to other crop systems. The usefulness of RADSeq as a tool for mapping of genetic loci will also be assessed by attempting to map polymorphisms associated with the self-incompatiblity loci of grasses. Identifying these genes is of high importance to grass breeders as they would allow greater control of mating during breeding programmes.

Technical Summary

RAD sequencing (RADSeq) is a high-throughput technique for marker discovery/genotyping. The technique generates short sequence reads adjacent to restriction enzyme sites, which can then serve as 'tags' to identify restriction polymorphisms between samples (similar to AFLP) or be mined to identify the presence of SNPs. By choosing a restriction enzyme which cuts infrequently within the study genome, the complexity of even large genomes can be reduced to a level where high-throughput marker discovery/genotyping can be performed using next-generation sequencing. A restriction enzyme is selected to produce 50-250,000 fragments per digest. Given that one lane of a Solexa flow-cell produces ~12 million reads, individual tags will be sequenced multiple times, making it possible to score presence/absence in the same manner as standard AFLP. However, RADSeq possesses the additional benefit of sampling sequence diversity around restriction sites, allowing for SNP discovery. Multiple individuals can be pooled within a single lane (using a unique 5bp molecular identifier for each sample) and the required coverage of tags needed for SNP genotyping will still be achieved. It is here that the power of next-generation sequencing becomes apparent: a whole flow-cell could potentially be used to assay an entire mapping population. We therefore aim to assess the feasibility of adapting the RADSeq technique to marker discovery/genotyping and mapping in crops, using the forage grass Lolium perenne as a model.We will initially test a series of restriction enzymes on the parental genotypes to produce two tag densities and identify parent-specific RADSeq markers, a subset of which will be validated using another genotyping technique. We will then conduct genotyping of a mapping population of 60 segregants and use this data to 1) construct a genetic map of RADSeq markers and 2) identify marker/trait associations for two genetic loci involved in self-incompatibility.

Planned Impact

1. Development of optimal wet-lab and bioinformatics protocols for implementation of RAD sequencing for NERC science 2a. Specific users this work might be of interest to and how they will benefit This proposal is aimed at deliveing much-improved technology for a wide range of BBSRC science researchers. The proposal details how we will prove the utility of and improve the accessibility of RADSeq technology. In particular we will develop robust wet-lab technologies and easy-to-use bioinformatics tools for RADSeq data generation and analysis. 2b. Techniques, methods or activities with which you will engage with this group We have already started a RADSeq users group within the UK and held two meetings. These have been well received, and well attended, and we intend to continue to promote RADSeq through this avenue. We also have a RADSeq wiki site, and one key goal is to develop this into a forum where RADSeq users (and newcomers) can find the information they need for starting, executing and delivering RADSeq analyses. 3a. Wider user interest The RADSeq technioque will be of interest we believe to a wide range of BBSRC-area science practitioners, many of whom may have decided against genetic analyses because of the perceived failings of existing technologies. 3b. Methods of disseminating information to this group We will enthusuiastically promote the RADSeq technology through presentation at research meetings, in publications and in online for a where RADSeq could be of interest to the community. 4. Milestones and measures of success In this 18 month grant, a key milestone will be the completion of the analyses of the differential utility of the different enzymes in the target plant genome. These will inform researchers planning to use RADSeq in the future; we are particularly interested in the performance of methylation sensitive enzymes in this regard. The delivery of optimised bioinformatic analytic tools will be the second major milestone. We will judge success in this segment by the take-up of the software by other users, and from the feedback we receive in its use. In the latter part of the project we will liaise closely with the staff of the GenePool, to ensure efficient transfer of the technology in to the GenePool portfolio. 5. Summary of resources We have requested funding for attendance of project staff at key UK science meetings. The RADSeq wiki is hosted (for free) on the University of Edinburgh computing services infrastructure.

Publications

10 25 50
 
Title Transmissions exhibition 
Description The Blaxter lab collaborated closely wth artists-in-residence (see http://www.ascus.org.uk/ciie-micro-residency-artists-announced/) in the Centre for immunity Infection and Evolution to inspire and be part of the final exhibition "Transmissions". Mark Blaxter appears in the film work produced by Anne Milne, and the work of the lab inspired Jo hodges and Robbie Coleman to produce a piece dedicated to the lab. 
Type Of Art Artwork 
Year Produced 2014 
Impact 'Transmissions' was showcased to the general public within a group exhibition 'Parallel Perspectives' in Summerhall as part of the Edinburgh International Science Festival 2015 art programme, How The Light Gets In . This exhibition of work susequently travelled LifeSpace, Dundee, returning to Edinburgh to showcase at the Tent Gallery, Edinburgh College of Art. 
URL http://www.ascus.org.uk/ciie-micro-residency-2/
 
Description RAD-Seq is an exciting technology that allows us to map and pin down rapidly genes that contribute to a wide array of different attributes of animals and plants. It can be used to identify genes that control traits such as production yield or disease tolerance.
Exploitation Route RAD-Seq is patented by KeyGene and commercial implementation is already well underway.
Sectors Agriculture, Food and Drink,Environment

 
Description BBSRC project grant (Striga)
Amount £43,647 (GBP)
Funding ID BB/J011703/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2015
 
Description BBSRC/DFID Project Grant (Ash Dieback)
Amount £162,746 (GBP)
Funding ID NORNEX 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 04/2015
 
Description Daphne Jackson Trust Fellowship
Amount £80,457 (GBP)
Organisation University of Surrey 
Department Daphne Jackson Trust
Sector Charity/Non Profit
Country United Kingdom
Start 08/2011 
End 08/2014
 
Description NERC Project Grant (RAD sequencing)
Amount £282,995 (GBP)
Funding ID NE/H019804/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 11/2010 
End 11/2011
 
Description NERC Project grant (Genome evolution)
Amount £469,000 (GBP)
Funding ID NE/J011355/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 09/2012 
End 09/2015
 
Description NERC NBAF 
Organisation Natural Environment Research Council
Country United Kingdom 
Sector Public 
PI Contribution We are a funded node of the NERC Biomolecular Analysis Facility. This collaboration involves three other University facilities.
Collaborator Contribution We are charged with delivering research support, sequencing and bioinformatics to NERC funded researchers.
Impact The collaboration is multi-disciplinary, involving genetics, genomics, bioinformatics, nmetabolomics and transcriptomics. The outputs include dozens of research papers and several collaborative grants.
Start Year 2006
 
Description Blaxter group - presentations and outreach 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The Blaxter group presented work at a wide range of national and international conferences, including PopGroup, the Arthropod Genomics Workshop, The C. elegans International Meeting, The Hydra Helminthology meeting, The European Society for Nematology, The UK Genome Science meeting, and others. At many of these venues, in addition to offering platform or poster presentations, we also presented workshops or training activities.
Year(s) Of Engagement Activity 2016
 
Description Blaxter group presentations and outreach 2015 
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 Mark Blaxter and research team communication and outreach 2015

Globodera genomics and blobtools software
25/02/2015 JHI Postgraduate Student Competition 2015 James Hutton Institute, Aberdeen, UK A tale of Two Peaks: Analysing Genomic Data from Potato Cyst Nematodes Talk
26/03/2015 JHI Cell and Molecular Sciences (CMS) seminar James Hutton Institute, Invergowrie, Dundee, UK Frustration and happiness : (De)-constructing parasite genomes Talk
16/06/2015 JHI Dundee effector consortium (DEC) meeting 2015 Birnam Arts and Conference Centre, Birnam, UK Variation within the Globodera pallida species complex: preliminary results Talk
03/09/2015 Molecular and Cellular Biology of Helminth Parasites IX Bratsera Hotel, Hydra, Greece Inter- and intra-specific analyses of the effector complement in potato cyst nematodes Poster
18/09/2015 UoE Postgraduate Poster Day University of Edinburgh, Edinburgh, UK Inter- and intra-specific analyses of the effector complement in potato cyst nematodes Poster
26/09/2015 Edinburgh University Doors Open Day University of Edinburgh, Edinburgh, UK Potato Cyst Nematodes (PCN) - Nematode parasites of potatoes Poster
30/11/2015 NextGenBug University of Edinburgh, Edinburgh, UK Blobtools: Blobology 2.0 Talk
01/12/2015 UK pollinator genomics meeting Roslin Institute, Edinburgh, UK Bees and Blobs Talk

LepBase
06/03/2015 EMARES Cambridge, UK The Bicyclus Genome Project Talk
06/03/2015 EMARES Cambridge, UK An introduction to Lepbase Talk
17/06/2015 Arthropod Genomics Manhattan, Kansas, USA Lepbase - A multi genome database for the Lepidoptera Poster
24/07/2015 10th Heliconius Meeting Gamboa, Panama Lepbase - A multi genome database for the Lepidoptera (API demonstration) Workshop
24/07/2015 10th Heliconius Meeting Panama Lepbase - A multi genome database for the Lepidoptera Poster
26/07/2015 10th Heliconius Meeting Panama Lepbase Workshop Talk
04/09/2015 Edinburgh Bioinformatics Edinburgh, UK Lepbase - A multi genome database for the Lepidoptera Talk
26/09/2015 Open Doors Day "Make a butterfly" interactive exhibition
26/09/2015 Edinburgh University Doors Open Day Edinburgh, UK Lepbase Multiple Sequence Alignments game Poster+Game
28/10/2015 NextgenBUG Dundee, UK Lepbase - an Ensembl (and more) for the Lepidoptera Talk

Nematode genomics
24.06.2015 20th International C. elegans Meeting Los Angeles USA A new evolutionary framework for the phylum Nematoda: a case study of HOX cluster evolution Poster
24.06.2015 20th International C. elegans Meeting Los Angeles USA Caenorhabditis Genomes Project Workshop (organiser and chair) Talk
24.06.2015 20th International C. elegans Meeting Los Angeles USA Current status of the CGP in Edinburgh Talk

Meloidogyne genomics
10-14 August 2015 ESEB Lausanne-Switzerland Genomic consequences of hybridization and the loss of meiotic recombination in Root-knot nematodes poster
15-18 December 2015 PopGroup Edinburgh-UK Genomic consequences of hybridization and the loss of meiotic recombination in Root-knot nematodes talk
23 February 2016 NextGenBug Edinburgh-UK Genomics of Root-knot nematodes talk
Year(s) Of Engagement Activity 2015
 
Description Blaxter lab workshops 
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 The Blaxter lab took our software products and research tools to various venues (Arthropod Genomics, UK Genome Science meeting, Butterfly Genomics) to present as workshops, training events or interactive sessions
Year(s) Of Engagement Activity 2016
 
Description Press releases and website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We have engaged actively with the University press office to promote press coverage of our research outcomes, particularly major publications (which have had coverage in national and international newspapers) and in blogs and other online media. We have also promoted major new initiatives such as additional core funding of the Edinburgh genomics facility.

Increased visibility of Edinburgh Genomics within the community; requests for comment by funders and government on matters pertaining to genomics.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016