The genomic basis of adaptation and species divergence in Senecio

Lead Research Organisation: University of Bristol
Department Name: Biological Sciences

Abstract

This project will exploit the latest developments in DNA sequencing and analysis technologies to study the genomic bases of adaptation and speciation in the plant genus Senecio (ragworts). The principal aim of this work is to determine the relative importance of differences in the coding and regulatory regions of genes in adaptation of species to contrasting environments, our null hypothesis being that these genomic regions contribute equally to adaptation and species divergence. To do this we will compare the nucleotide sequences of protein-coding and regulatory regions of the genomes of Senecio aethnensis and S. chrysanthemifolius, adapted to high and low altitudes, respectively, on Mt. Etna, Sicily, and a hybrid formed between them, S. squalidus (Oxford ragwort), which evolved in Britain following introduction of plants to Oxford from a hybrid zone on Mt. Etna 300 years ago. All 3 Senecio species are adapted to very different habitats and these phenotypic differences will be reflected by genotypic differences detectable by new comparative genomic technologies. Indeed, our previous NERC-funded research has revealed differences in gene expression between the 3 species correlated with phenotypic adaptation. This project will analyse these genetic differences further, and expand genomic comparison through an analysis of the entire gene-rich portion (protein-coding and regulatory sequences) of the genomes of the 3 species. To do this we will establish a partial reference genome by sequencing the gene-space, the non-repetitive fraction of the genome, of Senecio squalidus. This will allow identification of protein-coding and regulatory regions (e.g. promoters and microRNA binding sites). Protein-coding regions of genes can be identified relatively easily using bioinformatic approaches, but identification of regulatory regions requires additional analyses using new technologies designed to identify: i) RNA polymerase- and transcription factor-binding sites (chromatin immunoprecipitation [ChIP-] sequencing), ii) microRNAs (recently discovered ubiquitous gene-regulatory factors) and their binding sites. Once identified, these regions can then be sequenced extensively in multiple individuals of all 3 species to identify differences between them. Evolutionary genetic analysis will then be used to compare patterns of DNA polymorphism to detect 'footprints' of natural selection in protein-coding and regulatory regions that may have been involved in local adaptation and speciation, and also to further investigate the evolutionary history of the 3 Senecio species. In addition, we will investigate changes to promoter binding, DNA methylation (gene silencing) and microRNA expression/targeting between the 3 Senecio species that may be involved in adaptation. Finally, we will test (using plants grown at different UV-regimes) whether altered expression/regulation of candidate genes for local adaptation to high UV light (in high altitude S. aethnensis) is determined by species divergence or phenotypic plasticity. This project will break new ground in evolutionary genomics by being the first to: 1) use functional and evolutionary genetic analyses to analyse the entire gene-space of a plant to detect 'footprints' of selection correlated with adaptation and speciation; 2) explore the role of microRNAs in adaptation and speciation; 3) use large-scale ChIP-sequencing in a plant species. This work will therefore provide unprecedented advances in our current understanding of the role of genomic change in adaptation and speciation.

Publications

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Description This project has exploited the latest developments in nucleic acid sequencing (Next Generation Sequencing, NGS) and analysis technologies to study the genomic bases of adaptation and speciation in the plant genus Senecio (ragworts). The principal aim of this work was to determine the relative importance of differences in the coding and regulatory regions of genes in adaptation of species to contrasting environments, our null hypothesis being that these genomic regions contribute equally to adaptation and species divergence.
To do this we compared the nucleotide sequences of protein-coding and regulatory regions of the genomes of Senecio aethnensis and S. chrysanthemifolius, adapted to high and low altitudes, respectively, on Mt. Etna, Sicily, and a hybrid formed between them, S. squalidus (Oxford ragwort), which evolved in Britain following introduction of plants to Oxford from a hybrid zone on Mt. Etna 300 years ago. All 3 Senecio species are adapted to very different habitats and these phenotypic differences are reflected in genotypic (genomic) differences detectable by new comparative genomic technologies. Our previous NERC-funded research revealed differences in gene expression between these species correlated with phenotypic adaptation so our prediction was that changes in gene regulatory sequences (non-coding sequences) would be more important for adaptation and species divergence than changes to coding regions.
To facilitate this genomic comparison we originally intended to produce a partial reference genome for S. squalidus by sequencing the gene-space, i.e. the non-repetitive fraction of the genome. The purpose of this was to aid in the identification of protein-coding and regulatory regions (involved in the control of when and where genes are expressed). However, by taking full advantage of improvements in NGS DNA sequencing technology capacity that have occurred during the course of the project, we have actually been able to carry out far more extensive genomic sequencing than was originally envisaged (approximately 80 fold genome coverage comprising Illumina and 454 and PacBio sequence data for S. squalidus and 50 fold genome coverage of Illumina data for S. aethnensis and S. chrysanthemifolius). The financial savings from this approach have also allowed us to undertake further additional sequencing work, including whole genome sequencing of the other two species in our experimental system, RAD sequencing for genetic mapping and whole genome methylation studies (see below) that have served to increase the scope of this project considerably. The only down side of this 60-fold increase in data generated by the project has been in the increased time taken to cope with its analysis by a single RA bioinformaticist - hence the two no-cost extensions to the project.
The great plus side to all this is that we now have completed full draft genome sequences for S. squalidus, S. chrysanthemifolius and S. aethnensis. As expected these draft genomes are somewhat fragmented (N50 [the size of contiguous sequences which make up 50% of the assembled genome] of 18kbp for S. squalidus), but together they represent a far more useful and important genomic resource than the simple gene-space sequence for S. squalidus presented in our original objectives. Having a reference genome for each of the 3 species in this study is also extremely important for making full use of our additional sequencing experiments (see below). This has vital implications for our ability to analyse regulatory sequences in particular (which would have been poorly represented in the gene-space) and also provides an important additional resource for plant genomic science in general. Furthermore, more complete genomic sequences allow us to carry out additional analyses such as characterisation of the repeat elements of the genome and has also yielded complete chloroplast genome sequences, with mitochondrial genomes soon to follow.
Improvement of the reference genomes is an ongoing process, and includes development of a genetic map of Senecio using RAD (restriction associated DNA) sequencing, a reduced representation approach that allows the identification of thousands of variable genetic markers in a mapping population. Integration of the genetic map with the genome assembly will allow ordering of many genomic fragments and will also serve to increase contiguity. The genomes have been annotated using our extensive RNA-seq (a method used to sequence the RNA transcripts of genes that are being expressed) data and have identified over 26,000 gene models. As part of our goal to understand the transcriptional differences identified in these species, we are currently analysing the promoter sequences of these genes, especially those that we have identified as differentially expressed.
To further analyse the activity of promoter regions we successfully completed our objective of developing a chromatin immunoprecipitation sequencing technique (ChIP-seq) for genus Senecio. This technique (which is not trivial) uses antibodies to specifically target both transcriptionally active and promoter-bound forms of RNA polymerase, so that the associated DNA can be purified and analysed by high-throughput sequencing. This is a quantitative technique that, through the mapping of these sequences reads back to the corresponding reference genome, allows not only the identification and characterisation of transcriptionally active promoters, but also provides data on the degree of that activity that can be correlated with gene expression data. The ChIP technique has been optimised for Senecio and our antibodies of choice, and samples from 3 individuals each of the 3 Senecio species used in this project have been sequenced. Analysis of these data has revealed those sites in the genome to which RNA polymerase is bound at the time of sampling, and has been used to identify both promoter elements, and their differential usage between the species. Analysis of these data is onging.
Our investigation into the mechanisms of control of gene expression also includes an analysis of microRNAs (recently discovered ubiquitous gene-regulatory factors) and their binding sites. Sequencing of miRNA libraries from multiple individuals of all 3 species has now been completed, and characterisation of the miRNAs is complete using both this data and in silico approaches through interrogation of the reference genomes. As well as previously characterised miRNA sequences, novel ones have also been identified in each species, especially (and of particular interest) in the hybrid derived S. squlaidus. These sequences have also been analysed to identify putative target genes for regulation. Additionally, sequence and expression level differences between individuals and species have been identified, as a first step in the investigation of epigenetic (epigenetics is the study of gene regulatory mechanisms that lie above the gene sequence itself) factors in Senecio. This epigenetics approach has been further enhanced by low coverage whole genome bisulfite sequencing of all three species to identify patterns of DNA methylation, a chemical modification of cytosines which is known to be important in regulating gene expression, and represents an important additional level of detail in our study of the underlying mechanisms regulating gene activity.
Evolutionary genetic analysis has now been used to compare patterns of DNA polymorphism to detect 'footprints' of natural selection in protein-coding and regulatory regions that may have been involved in local adaptation and speciation, and also to further investigate the evolutionary history of the 3 Senecio species. In addition, we are investigating changes to promoter binding, DNA methylation (gene silencing) and microRNA expression/targeting between the 3 Senecio species that may be involved in adaptation. Integration of these diverse datasets is ongoing in preparation for the submission of a substantial genome analysis paper to a high impact journal - our target is Nature Genetics.
All the experimental objectives of this project are now complete and have generated huge amounts of additional sequence data that goes way beyond that envisaged in the original objectives of the proposal. The completion of three reference genomes vastly exceeds our initial goal of a single gene space reference, and is proving to be an very valuable resource for us and our collaborators. Analysis of these data is ongoing, and is focussed upon integrating the different experimental approaches to provide a coherent picture of the processes involved in regulation of gene expression, while also providing an important new genomic resource in the genome sequences of three Senecio species. This project has broken new ground in evolutionary genomics by being the first to: 1) use functional and evolutionary genetic analyses to analyse the entire gene-space of a plant to detect 'footprints' of selection correlated with adaptation and speciation; 2) explore the role of microRNAs and DNA methylation in adaptation and speciation; 3) use large-scale ChIP-sequencing targeted to promoters in a plant species. This work will therefore provide unprecedented advances in our current understanding of the role of genomic change in adaptation and speciation.
Exploitation Route The story of Oxford ragwort's invasion of the UK via railway lines during the industrial revolution and its subsequent hybridization with native groundsel to generate three new Senecio taxa in the UK is of perennial interest to naturalists and anyone with an interest in the British flora. SJH has been invited to talk about the broad aspects of this research many times by local naturalist trusts and groups, e.g. Bristol Naturalists Trust, Avon and Somerset Wildlife Trust, Friends of the University of Bristol Botanic Garden. The major beneficiaries of this research will be academics and researchers. A major output will be the draft genome of Senecio squalidus, an important genomic resource that will integrate all of our sequencing data into a publicly accessible resource for use by the wider scientific community. This will facilitate comparative genomics studies, as well as being of particular interest to those working on Senecio species, and the Asteraceae in general, by providing a reference genome. For example, this project has lead to collaborative work with a French research group in Lille studying self-incompatibility mechanisms in chicory, by providing genomic resources that allow for syntenic analyses (based on the conservation of genes and order of loci on chromosomes between species) to aid in the identification of the genetic elements responsible for this phenomenon. This demonstrates the potential of this work to be applied to related species, which includes crop plants and has implications for commercial breeders as well as academic use. In Bristol, our analysis of microRNAs has also lead to a collaboration with Professor Philip Donoghue in Earth sciences, using microRNA sequences to improve and resolve deep phylogenetic relationships. Another immediate advantage has been that we have been able to begin investigation of epigenetic mechanisms (regulatory processes which arise from mechanisms distinct from the underlying DNA sequence) in Senecio, in the context of local adaptation and speciation, a novel and important approach that promises to yield novel insights into the role of these recently discovered systems and which has formed the basis of future grant funding applications.



Our overall goal of determining the relative importance of coding and regulatory genetic elements in adaptation to contrasting environments will be of great interest to evolutionary biologists. It has implications for the understanding of local adaptation, speciation and species range expansion - the latter of which is a particular area of investigatiom for scientists working to understand the effects of climate change on populations. Through our use of a hybrid model system, our results will also provide insights into the genomic changes that accompany hybridisation and will therefore serve as a resource for this wider research community.



Additionally, our development of molecular methods, especially our ChIP-seq approach, will be of great use in expanding experimental approaches available for Senecio species. It is also important to note that we have undertaken a wide range of next generation sequencing approaches in a non-model organism, derived from a wild population. To date, the majority of genomics work has focused on model organisms due to the relative simplicity afforded by systems which are already well characterised at the molecular level. However, many of these model systems are of limited interest in an evolutionary and ecological context. Our demonstration that these techniques are applicable to less well characterised experimental systems is an important confirmation of the wider usefulness of these approaches.
Sectors Environment

 
Description Impact has been purely scientific.
First Year Of Impact 2011
Sector Education,Environment,Other
 
Description New Phytologist Trust Editors Grant
Amount £10,000 (GBP)
Organisation New Phytologist 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 01/2019
 
Description University of Oxford start up award
Amount £150,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 10/2019
 
Title SenecioDB 
Description SenecioDB is an expanding genomics resource for plant scientists, particularly those working on ragworts (Senecio) and the daisy family (Asteraceae) more generally. SenecioDB was initially developed under NERC grant NE/F001207/1, and has been expanded and updated under the current research project. It presently incorporates a database of over 100,000 ESTs and contigs derived from floral and leaf tissue of five Senecio taxa. By following the menu links, researchers will be able to query this database with their own sequences or by keyword. Bioinformatics tools are also available to mine the EST data for markers such as SNPs and SSRs. All ESTs have been compared to Asteraceae sequences from the Compositae Genome Project and links/alignments are available where matches have been identified. Further background information on Senecio and our work is also hosted on the site. In addition, contigs from recently completed whole transcriptome Illuina sequencing of 6 tissues from Senecio squalidus is presently being prepared for inclusion. 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? Yes  
Impact SenecioDB represents the most complete collection of Senecio sequencing data available, and is thus an invaluable resource for researchers with an interest in this genus as well as for scientists with an interest in teh Asteraceae more generally. IT is constantly expanding, and will serve as the main portal for release of the Senecio draft genomes on publication. 
URL http://www.seneciodb.org/wordpress/
 
Description Compositae Genome Project 
Organisation University of California, Davis
Country United States 
Sector Academic/University 
PI Contribution The genomes of Senecio squalidus, Senecio aethnensis and Senecio chrysanthemifolius represent an important addition to the US basd Compositae genome project. Unpublished data was also used for syntenic analysis with the (at the time) unpublished Lactuca sativa genome project.
Collaborator Contribution Collaboration with Prof Michelmore and the Compositae Genome Project has provided unprecedented access to unpublished genomic resources (contig scaffolds, BAC sequences, markers) for lettuce that are greatly facilitating assembly of the Senecio genome.
Impact Us eof unpublished genomic resources from Lactuca sativa have been used for syntenic analysis and assembly of the Senecio genome.
Start Year 2011
 
Description Continued collaboration with Dr Adrian Brennan 
Organisation Durham University
Country United Kingdom 
Sector Academic/University 
PI Contribution Hiscock has co-written some papers with Dr Brennan based on his mapping data generated on a NERC grant to Richard Abbott on which Hiscock was also a Co-I. Some of these mapping data have been used to help with our genome assemblies.
Collaborator Contribution Dr Brennan has provided DNA samples from his Senecio mapping population that we used in our RAD-seq analysis. Dr Brennan has also helped and advised with other analyses.
Impact Brennan AC, Hiscock SJ, Abbott RJ (2014) Interspecific crossing and genetic mapping reveal intrinsic genomic incompatibility between two Senecio species that form a hybrid zone on Mount Etna, Sicily. Heredity, in press. Brennan AC, Barker D, Hiscock SJ, Abbott RJ. (2012) Molecular genetic and quantitative trait divergence associated with recent hybrid speciation: a study of Senecio squalidus (Asteraceae). Heredity 108: 87-95.
Start Year 2008
 
Description Epigenetic basis of speciation in African lake cichlid fishes 
Organisation University of Bristol
Department School of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I am co-I on this EU Marie Currie Fellowship Award
Collaborator Contribution My collaborators are Dr Martin Genner (PI) and Tom Batstone (Bioinformatician). Both discuss and advise on my Senecio work and Tom continues to contribute to bioinformatic analyses of the Senecio genome
Impact Too early for outcomes
Start Year 2015
 
Description Genomic repeat analysis with Professor Andrew Leitch 
Organisation Queen Mary University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided Illumina genome sequences for whole genome repeat analysis using repeat explorer, and have continued this analysis across Senecio squalidus, aethnensis and chrysanthemifolius, to identify and classify the repeat elements present in these genomes.
Collaborator Contribution Professor Andrew Leitch and his reserach group have extensive expertise in this type of analysis, and have provided training for RA Tom Batstone in it's use. In addition, they have advised on the optimal approaches to use and have helped with follow on analysis of identified repeat clusters.
Impact The proportion of each of our three genomes of interest that is composed of repeat elements has been determined. Additionally, these repeats have been classified, aiding in genome annotation. Follow on studies examining repeat variability and their potential impact between these species are presently underway.
Start Year 2013
 
Description Genomic, transcriptome and RAD sequencing of Senecio species 
Organisation Aberystwyth University
Country United Kingdom 
Sector Academic/University 
PI Contribution We supplied genomic DNA and total RNA from Senecio squalidus, Senecio aethnensis and Senecio chrystanethemifolius for Illumina sequuencning. Additionally, we provided genomic DNA from a mapping population of Senecio squalidus for RAD sequencing. RA Tom Batstone was also involved in the preparation of RAD samples and library construction. Data analysis of genomic and transcriptome was carried out by ourselves.
Collaborator Contribution Sequencing services were supplied with a mixed model of service provision and in kind contribution by Dr Matt Hegarty. Training of RA Tom Batstone in teh construction of RAD sequencing libraries and Nextera sequencning libraries was also provided. Additionally, analysis of RAD sequencing data was carried out by Dr Matt Hegarty.
Impact This has resulted in the draft genome sequences of Senecio chrysanthemifolius and Senecio aethnensis. Additionally, RNA-seq data from six tissues of Senecio squalidus has been used for annotation of the squalidus genome. RAD sequencing data has been used for the construction of genomic maps to which the squalidus genome assembly has been anchored.
Start Year 2010
 
Description Grant application to NERC: NE/G017646/1 
Organisation University of Oxford
Department Department of Plant Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborative research application involving Hiscock and Abbott and new collaborator Dr Dmitry Filatov (Department of Plant Sciences, University of Oxford)
Collaborator Contribution Hiscock, Filatov, Abbott and Hegarty Jointly wrote a grant application to NERC - 'The genomic basis of adaptation and species divergence in Senecio. The award was funded as a joint award to Bristol and Oxford - NE/G018448/1 and NE/G017646/1.
Impact The collaborative research proposal was funded as a joint award to Bristol and Oxford - NE/G018448/1 and NE/G017646/1.
Start Year 2009
 
Description Hybrid and polyploid speciation in Sorbus 
Organisation Royal Botanic Gardens, Kew
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution I am a co-I on this project lead by Dr Mike Fay - I contribute genetic and population genetic advice and assistance
Collaborator Contribution My Kew partners have provided the funding and the post doc works in the Jodrell Laboratory
Impact No outputs yet
Start Year 2015
 
Description Research collaboration with chicory researchers at the University of Lille 
Organisation University of Lille
Country France 
Sector Academic/University 
PI Contribution Prof Hiscock visited the lab of Prof Theo Hendriks and Dr Marie-Christine Quillet (INRA, University of Lille) in France to discuss collaboration. Prof Hendriks' group is mapping and sequencing the chicory genome. This resulted in mutual access to unpublished sequence data that is now greatly facilitating assembly of the Senecio and chicory genomes through synteny. It has also spawned a collaborative project to map and sequence the self-incompatibility (S) locus of Senecio and chicory. An MSci student has also just been allocated to continue investigating this data in Bristol.
Collaborator Contribution Collaborators have provided genomic and transcriptomic sequence data for the chicory S-locus region, thereby allowing us to identify syntenic regions in the Senecio genome.
Impact Collaboration resulted in submission of a research proposal to BBSRC - application unsuccessful.
Start Year 2011
 
Description miRNA classification for phylogenetics 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided genomic and miRNA sequencing data from the Senecio genome project for analysis by Professor Phil Donoghue and his group, who are working on resolution of deep phylogenies using miRNAs. This led to additional low coverage genome sequencning and de novo assembly as well as small RNA sequencning for a wide variety of plant species. Additionally, we have provided expertise in molecular, sequencing and data analysis techniques including training of group members.
Collaborator Contribution Professor Phil Donoghue and his group have provided expertise in the identification and classification of miRNAs, through the development of custom computational pipelines which has contributed to both the analysis of Senecio miRNAs in this grant and other plant species of interest.
Impact miRNAs, including 27 previously uncharacterised, from our three Senecio species of study have been identified and characterised. DIfferential expression has also been analysed between and within species and putative target genes identified.
Start Year 2011