14 ERA-CAPS: INvestigating TRiticeae EPIgenomes for Domestication (INTREPID)

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

Plant breeding uses DNA sequence variation to make new allelic combinations for crop improvement. Our creation of the first wheat gene sequence assemblies (Brenchley et al Nature 491, 705) has enabled new levels of high throughput precise genotyping for breeding this globally important crop. Nevertheless, there are other levels of heritable variation, such as epigenetic modifications, that are widely thought to play a key role in shaping genomes and creating new variation. We have recently developed highly efficient re-sequencing technologies for wheat that can measure DNA methylation in genes of multiple lines. This provides an outstanding opportunity to assess epigenetic variation in a major polyploid crop and understand how it may influence traits. The overall objective of this proposal is to use newly available wheat genome resources, together with our innovative application of exome capture and bisulphite sequencing, to measure epigenetic modifications in wheat genes, and relate these to gene expression and the acquisition of new phenotypes, and how they may contribute to genetic changes such as gene loss during polyploid formation.

Technical Summary

The production of new hybrids is an important way of improving crops as they exhibit novel traits directly after hybrid formation, which are not found in progenitor parents. Growing evidence points to possible epigenetic origins for these emergent phenotypes. The scale and heritability of epigenetic modifications therefore needs to be measured, related to potential changes in gene and chromosome function and then taken into account in breeding as a source of variation in breeding.
Here, we aim to build on our collective experience in plant epigenetics and genomics to map the epigenome of bread wheat. Outputs of this project will be of immediate value for breeders for understanding the extent and contribution of epi-allelic variation to traits and in the choice of parental epi-allelic variation in making new hybrids. The project will also exploit experimental advantages of wheat to understand how epigenetic marks are re-programmed during the formation of new wheat hybrids, and how their independently maintained genomes influence each other during stabilization of the new hexaploid genomes. We have established four key foundations for mapping and understanding the wheat epigenome: the first genome sequence assembly of wheat; an efficient method for the cost-effective sequencing of the gene space of multiple wheat genomes and for determining genome-wide DNA methylation patterns an improved understanding of the mechanisms of epigenetic inheritance and evidence of altered gene expression in wheat hybrids.
This will generate new knowledge of how epi-alleles are formed and maintained, how the genomes of polyploid wheat influence each other, and how they influence gene function. It will have an important impact on wheat breeding by establishing the extent of epigenetic variation in wheat lines and its consequences on genome function and predicted phenotypes. Such information can guide the choice of parents for hybrid formation and explain aspects of missing heritability.

Planned Impact

Wheat is one of the three global crops providing the bulk of human nutrition. Large-scale coordinated research programmes are aiming to increase yields and reduce agricultural inputs in order to meet future consumption patterns and to mitigate the predicted effects of a changed growing climate. This project will have a fundamentally important impact on wheat breeding by understanding, for the first time, the extent of inherited epigenetic variation in wheat lines, and its consequences on genome function and predicted phenotypes. Such variation has been predicted to contribute to creating new phenotypes and heterotic yield increases in hybrids, but it has not yet been assessed at a genome scale in wheat. Such variation may explain aspects of missing or low heritability and could be used in breeding programmes. The project will show how new epigenetic variation is generated in hybrids, how such variation is stabilized, how new patterns of gene expression are created and stabilized; and how patterns of epi-alleles and traits can be influenced by the environment. This can guide crop improvement strategies by determining the choice of parents for hybrid formation, and by identifying subsequent epigenetic variation and measuring its stability across generations before incorporation into breeding programmes. In addition to these basic and applied outputs of this project, we will develop bioinformatic tools for identifying and analysing epigenetic marks and tracking their potential phenotypic consequences through gene expression network analyses. These impacts will be delivered through the open cyber-infrastructure provided by iPlant and through databases such as Ensembl genomes, through close engagement with breeders, and through publications and training programmes.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/N005104/1 01/10/2015 30/09/2016 £425,122
BB/N005104/2 Transfer BB/N005104/1 01/10/2016 30/03/2019 £309,763
 
Description First description of the wheat epigenome:
• Developed technology/methodology advance combining sequence capture, bisulfite treatment and genome specific assignment of reads using homoeologous SNPs
• Analysed genome wide methylation patterns including reports of genome specific methylation and conserved methylation patterns across the three genomes
• Associating methylation of a single genome with genome specific gene expression
• shown Non-CpG methylation is enriched in sub-genome specific methylation suggesting a potential association of sub-genome specific methylation with pseudo genes
• Shown that tri-genome methylation is highly conserved with a diploid wheat progenitor while sub-genome specific methylation shows more variation i.e. context specific variation.

Population epigenomic diversity across the A. E. Watkins bread wheat landrace cultivar collection

There is a high level of genetic diversity in the A. E. Watkins bread wheat landrace collection and, we predict, also a high level of epigenetic diversity. This represents a dual source of variation not used in modern breeding that can be exploited for wheat improvement. Here, we analyze genotype and DNA methylation, an important mechanism of epigenetic gene expression control that can be passed between generations, across a core set of the Watkins collection. This core set includes 105 lines and captures the majority of the diversity in the Watkins collection. Using sodium bisulfite treatment and targeted gene enrichment we can survey genome-wide methylation and genotype across the three sub-genomes of allohexaploid wheat.

Using a 12Mb capture probe set we were able to analyze an average of 48 Mb of the wheat genome per sample at a minimum depth of 10X. Methylation shows high variability within the Watkins collection with 42.1% of analyzed sites classified as single methylation polymorphism sites, or SMPs, between the samples. The vast majority of these SMPs (91.5%) are rare variants that were seen in less than 10% of the samples. Tri-genome methylation is more conserved between accessions than genome specific methylation and therefore likely to be the most stable form of methylation. Genome specific methylation sites show enrichment for homoeologous SNPs that differentiate the genome that is methylated from the other two sub-genomes and this SNP typically infers a CpG site from a non-CpG site.

We have observed that methylation is a standalone source of variation in the absence of genetic variation, however, it is clear that if two wheat accessions show more closely related genotypes then their methylomes are more likely to be related. Both methylation and genotype are clearly influenced by the geographical origin of the sample, although it appears that genotypic profiles cluster across wider geographic regions while the methylation profiles of accessions tend to cluster into more local groups. These lines clustering locally by methylation may be more likely to have evolved with similar environmental conditions for growth and this may have influenced their methylation profiles. Therefore, we hypothesize that methylation acts as a fast-adaptive response to environmental stimulus that may later become 'hard coded' as SNPs.

We are currently collaborating with Klaus Mayer's team in Munich to assess the gene networks that are targeted by methylation across the Watkins collection and how these vary between accessions. Furthermore, we are analyzing RNA-seq data for 12 of the samples from the Watkins collection to allow correlation of genotype, methylation and gene expression in this analysis. Finally, we are working with Cold Spring Harbor laboratories to sequence the full methylome of Chinese Spring wheat.
Exploitation Route Crop breeding is driven by genetic variation. It is likely that epigenetic variation significantly contributes to the phenotypic variation for some of the key traits breeders are interested in. We have already established that a huge amount of epi-genetic variation can be seen in a diverse global collection of bread wheat landraces. While this is of fundamental biological interest, it is not useful to wheat breeders. Our new EAGER proposal following on from INTREPID will turn this fundamental discovery into a breakthrough technology. To achieve this over the past few months we have worked with breeders to identify: genes and pathways controlling key traits relevant to them; a panel of relevant material and a clear understanding of how an assay could be developed that would integrate into existing genotyping pipelines. The EAGER project will also address many of the questions breeders are asking. Is there epigenetic variations in genes controlling traits they are interested in? How stable is the epigenetic variation? How is it inherited? Can I just follow a haplotype with existing SNP markers or are there methylated and unmethylated versions of the same haplotype. This EAGER project will address this questions and deliver them a KASP based assay to track epi-alleles.
Sectors Agriculture, Food and Drink

 
Description SEQUENCING THE GENIC PORTION OF SEEDS OF DISCOVERY ADVANCE PRE- BREEDING GERMPLASM TO UNCOVER THE GENETIC VARIATION
Amount £330,000 (GBP)
Funding ID BBS/OS/NW/000017 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 10/2019
 
Title Methyl-seq methods for wheat 
Description Background: Bread wheat has a large complex genome that makes whole genome resequencing costly. Therefore, genome complexity reduction techniques such as sequence capture make re-sequencing cost effective. With a high- quality draft wheat genome now available it is possible to design capture probe sets and to use them to accurately genotype and anchor SNPs to the genome. Furthermore, in addition to genetic variation, epigenetic variation provides a source of natural variation contributing to changes in gene expression and phenotype that can be profiled at the base pair level using sequence capture coupled with bisulphite treatment. Here, we present a new 12 Mbp wheat capture probe set, that allows both the profiling of genotype and methylation from the same DNA sample. Furthermore, we present a method, based on Agilent SureSelect Methyl-Seq, that will use a single capture assay as a starting point to allow both DNA sequencing and methyl-seq. Results: Our method uses a single capture assay that is sequentially split and used for both DNA sequencing and methyl-seq. The resultant genotype and epi-type data is highly comparable in terms of coverage and SNP/methylation site identification to that generated from separate captures for DNA sequencing and methyl-seq. Furthermore, by defining SNP frequencies in a diverse landrace from the Watkins collection we highlight the importance of having genotype data to prevent false positive methylation calls. Finally, we present the design of a new 12 Mbp wheat capture and demonstrate its successful application to re-sequence wheat. Conclusions: We present a cost-effective method for performing both DNA sequencing and methyl-seq from a single capture reaction thus reducing reagent costs, sample preparation time and DNA requirements for these complementary analyses. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact As yet not known 
 
Title promoter exon capture platform 
Description Whole genome shotgun re-sequencing of wheat is expensive because of its large, repetitive genome. Moreover, sequence data can fail to map uniquely to the reference genome making it difficult to unambiguously assign variation. Re-sequencing using target capture enables sequencing of large numbers of individuals at high coverage to reliably identify variants associated with important agronomic traits. Previous studies have implemented cDNA/exon or gene-based probe sets where promoter and intron sequence is largely missing alongside newly characterized genes from the recent improved reference sequences. Results We present and validate two gold standard capture probe sets for hexaploid bread wheat, a gene and a putative promoter capture, which are designed using recently developed genome sequence and annotation resources. The captures can be combined or used independently. We demonstrate that the capture probe sets effectively enrich the high confidence genes and putative promoter regions that were identified in the genome alongside a large proportion of the low confidence genes and associated promoters. Finally, we demonstrate successful sample multiplexing that allows generation of adequate sequence coverage for SNP calling while significantly reducing cost per sample for gene and putative promoter capture. Conclusions We show that a capture design employing an 'island strategy' can enable analysis of the large gene/putative promoter space of wheat with only 2x160 Mb probe sets. Furthermore, these assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Currently being used by DFW, CIMMYT and US CAPS projects 
 
Title A genome-wide DNA methylation survey of hexaploid wheat 
Description DNA methylation is an important mechanism of epigenetic gene expression control that can be passed between generations. Here, we use sodium bisulfite treatment and targeted gene enrichment to study genome-wide methylation across the three sub-genomes of allohexaploid wheat. While the majority of methylation is conserved across all three genomes we demonstrate that differential methylation exists between the sub-genomes in approximately equal proportions. We correlate sub-genome specific promoter methylation with decreased expression levels and show that altered growing temperature has a small effect on methylation state, identifying a small but functionally relevant set of methylated genes. Finally, we demonstrate long-term methylation maintenance using a comparison between the D sub-genome of hexaploid wheat and its progenitor Aegilops tauschii. We show that tri-genome methylation is highly conserved with the diploid wheat progenitor while sub-genome specific methylation shows more variation. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Differential methylation between homologous genomes in wheat, evidence for the stability of methylation in the genie portion of wheat 
URL https://www.ebi.ac.uk/ena/data/view/PRJEB8762
 
Title Epigenomic variation across a polyploid wheat diversity collection 
Description We survey genotype and DNA methylation across the core Watkins bread wheat landrace collection. We observe high transposable element variability and expansion, most frequently in retrotransposons, alongside high epigenomic diversity; while there is an association between methylation and genotype, methylation is a standalone source of variation between closely related accessions. Both methylation and genotype are influenced by geographic origin. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Lead to an EAGER grant to translate the research 
URL https://www.ebi.ac.uk/ena/data/view/PRJEB23320
 
Title Watkins core collection re-sequencing data 
Description Re-sequence data for the Watkins collection 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact International collabration 
URL https://grassroots.tools/data/under_license/toronto/
 
Title promoter exon capture data 
Description The data contain the design space for the promoter and exon used in our capture 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Whole genome shotgun re-sequencing of wheat is expensive because of its large, repetitive genome. Moreover, sequence data can fail to map uniquely to the reference genome making it difficult to unambiguously assign variation. Re-sequencing using target capture enables sequencing of large numbers of individuals at high coverage to reliably identify variants associated with important agronomic traits. We present two gold standard capture probe sets for hexaploid bread wheat, a gene and a promoter capture, which are designed using recently developed genome sequence and annotation resources. The captures can be combined or used independently. The capture probe sets effectively enrich the high confidence genes and promoters that were identified in the genome alongside a large proportion of the low confidence genes and promoters. We use a capture design employing an 'island strategy' to enable analysis of the large gene/promoter space of wheat with only 2x160 Mb NimbelGen probe sets. Furthermore, these assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations. Here, we release the targeted sequence of the capture probe sets on the wheat RefSeqv1, the design space that was used to tile our capture probes across and finally the positions of the probes themselves across this design space for both the gene and promoter capture probe sets. 
URL https://opendata.earlham.ac.uk/wheat/under_license/toronto/Gardiner_2018-07-04_Wheat-gene-promoter-c...
 
Description CIMMYT seeds of discovery 
Organisation Australian National University (ANU)
Country Australia 
Sector Academic/University 
PI Contribution Collaboration to provide Advance genetic approaches
Collaborator Contribution Access to wheat material and phenotyping data
Impact None yet
Start Year 2016
 
Description CIMMYT seeds of discovery 
Organisation International Centre for Maize and Wheat Improvement (CIMMYT)
Country Mexico 
Sector Charity/Non Profit 
PI Contribution Collaboration to provide Advance genetic approaches
Collaborator Contribution Access to wheat material and phenotyping data
Impact None yet
Start Year 2016
 
Description EAGER - Eduard Akhunov 
Organisation Kansas State University
Country United States 
Sector Academic/University 
PI Contribution Joint funding and publications, method development
Collaborator Contribution Access to material and bioinformatics methods
Impact EAGER -BBSRC grant A PATHWAY TO THE EXPLOITATION OF EPIGENETIC VARIATION IN UK, US AND INTERNATIONAL BREEDING PROGRAMMES Gardiner, L.-J., Brabbs, T., Akhunov, A., Jordan, K., Budak, H., Richmond, T., et al. (2019). Integrating genomic resources to present full gene and putative promoter capture probe sets for bread wheat. GigaScience. http://doi.org/10.1093/gigascience/giz018 IWGS Consortium, Jordan, K. W., Wang, S., Lun, Y., Gardiner, L.-J., MacLachlan, R., et al. (2015). A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes. Genome Biology, 16(1), 680. http://doi.org/10.1186/s13059-015-0606-4
Start Year 2018
 
Description IBM 
Organisation IBM
Department IBM UK Ltd
Country United Kingdom 
Sector Private 
PI Contribution This project aims to leverage these new resources together with large gene expression data sets to reconstruct regulatory networks controlling important traits. With outputs feeding directly into UK and International wheat research and UK wheat breeding programmes. The collaboration focuses around two large data sets: the first, a high resolution time series data set for wheat and the second, a developmental time course for 16 international elite cultivars. The project aims to: 1. Develop normalisation methods for the transcriptome data sets (RNA-seq data), specific for dealing with the complexities of time course data and a complex polyploid genome. Benchmark these new approaches against existing tools. 2. Develop AI pattern matching algorithms to identify gene-expression modules and correlate these with biological processes. Use pattern matching algorithms to interrogate the high resolution data sets and to identify differences between regulatory networks in elite cultivars 3. Correlate changes in temporal or developmental expression patterns with changes in promoter architecture 4. Use network inference, co-expression and gene modules to reconstruct regulatory networks. Identify how network structure changes over time and between cultivars. This project will have impact for scientists as it will address fundamental questions about temporal regulation of processes in wheat and how a complex networks work across multiple genomes within a polyploid. By comparing networks across elite cultivars it will also have an impact for UK and international wheat breeders, identification of how breeding programmes have changed the architure of regulatory networks. This will have important impact for the selection of future networks for breeders to target or for assessing the selection of breeding material. It will allow breeders to make full use of the new resources and technologies. To ensure the impact to the UK industry is realised we aim to present this work a UK networking meeting and to visit and involve three private UK breeding companies RAGT, KWS and Elsoms seeds in early output from the project.
Collaborator Contribution IBM have are involved in applying ML and AI approaches to analyse gene expression data in wheat and reconstruct gene networks.
Impact No outputs to data
Start Year 2017
 
Description INvestigating TRiticeae EPIgenomes for Domestication 
Organisation Cold Spring Harbor Laboratory (CSHL)
Country United States 
Sector Charity/Non Profit 
PI Contribution Development of approaches to sequence the epigenome and bioinformatics pipelines
Collaborator Contribution access to material and expertise in systems genomic and bioinformatics.
Impact not yet
Start Year 2015
 
Description INvestigating TRiticeae EPIgenomes for Domestication 
Organisation Helmholtz Zentrum München
Country Germany 
Sector Public 
PI Contribution Development of approaches to sequence the epigenome and bioinformatics pipelines
Collaborator Contribution access to material and expertise in systems genomic and bioinformatics.
Impact not yet
Start Year 2015
 
Description IWYP 
Organisation Australian National University (ANU)
Department Division of Plant Sciences
PI Contribution My group lead this work, we are involved in the genotyping and bioinformatics
Collaborator Contribution The three other groups are involved in generating the field phenotyping and plant physiology measurements.
Impact none yet
Start Year 2016
 
Description IWYP 
Organisation International Centre for Maize and Wheat Improvement (CIMMYT)
Country Mexico 
Sector Charity/Non Profit 
PI Contribution My group lead this work, we are involved in the genotyping and bioinformatics
Collaborator Contribution The three other groups are involved in generating the field phenotyping and plant physiology measurements.
Impact none yet
Start Year 2016
 
Description IWYP 
Organisation Lancaster University
Department Lancaster Environment Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution My group lead this work, we are involved in the genotyping and bioinformatics
Collaborator Contribution The three other groups are involved in generating the field phenotyping and plant physiology measurements.
Impact none yet
Start Year 2016
 
Description KWS 
Organisation KWS UK
Country United Kingdom 
Sector Private 
PI Contribution Generated double haploid population for the IWYP project and provide material for INTREPID and the BBSRC/EAGER work
Collaborator Contribution Know of the techniques and approaches we are using, early access to the data we generate
Impact Double haploid seed population
Start Year 2017
 
Description Moussa Benhamed 
Organisation University of Paris-Saclay
PI Contribution WE are collaborating with Moussa Benhamed to develop Next generation sequencing technology to explore the 3D structural organisation in the wheat nucleus and epigenetic regulation in wheat.
Collaborator Contribution The have built sequencing libraries for design future wheat and are providing staff training
Impact Production of sequencing libraries analysis of histone modification, 3D genome structural organisation and Chromatin accessibility.
Start Year 2018
 
Description RAGT 
Organisation RAGT Seeds
Country United Kingdom 
Sector Learned Society 
PI Contribution Identify key material for our EAGER project and Design future wheat. Support for collaboration with IBM.
Collaborator Contribution Discussion of methods and analysis of material relevant to RAGT.
Impact none yet
Start Year 2018
 
Description Oral presentation and poster presentation at the Plant and Animal genome conference (PAG) San Diego 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk entitled "Exploring epigenetic diversity in polyploid wheat" to an audience of scientists including; PIs, postdocs, students, industrial partners and breeders. This highlighted the findings of our epigenetic diversity study.
Year(s) Of Engagement Activity 2018
URL https://pag.confex.com/pag/xxvi/meetingapp.cgi/Session/4886
 
Description Oral presentation at conference: Training on Durum Wheat Genomics and Breeding, University of Bologna, Italy 
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 Oral presentation at training conference to educate and train breeders, farmers and students in bioinformatic techniques for the analysis of wheat genomics data
Year(s) Of Engagement Activity 2017
URL http://www.wheatinitiative.org/events/durum-ewg-workshop-bioinformatics-advance-wheat-breeding
 
Description Oral presentation at the Wheat and Barley Legacy for breeding improvement annual meeting at the University of Haifa, Israel 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attended and presented work at the Wheat and Barley Legacy for breeding improvement annual meeting at the University of Haifa, Israel. This built links with researchers in Italy, France and Israel for future collaboration and sparked their interest in epigenetic research.
Year(s) Of Engagement Activity 2017
 
Description Oral presentation: Agilent User group meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact I presented my work analysing the methylation of wheat to many of the users of Agilent technology in the UK since I implemented Agilent technology for the study. Many PI's and postdoctoral researchers were present and were interested in how they could use this methodology for their own analyses.
Year(s) Of Engagement Activity 2018
URL https://www.chem.agilent.com/edm/2017/06/Uk_Seminar_2017/Documents/GenUGM/Agenda_GUGM.pdf
 
Description Talk and discussion with Elsoms seeds 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact The talk was to breeders and crop scientist work at Elsoms seed, The aim was to raise awareness of the work that Design future wheat and my group were doing. This lead to a letter of support for our BBSRC grant and a line of communication with their wheat and brassica breeders.
Year(s) Of Engagement Activity 2018