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

Lead Research Organisation: John Innes Centre
Department Name: Crop Genetics

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

Please refer to the Lead Applicant JeS form.
 
Description Work is progressing well. Initial analyses suggest a prominent role for epigenetic variation in the adaptation of wheat landraces to growth in different environments. Progress in analysing differences between the D genomes of diploid progenitor and hexaploid wheat are advanced

we have shown that gene expression in the diploid context (the Ae tauschii DD genome) is reduced in the hexaploid AABBDD genome context. This was validated by quantitative methods and we showed that chromatin compaction occurs that is known to reduce gene expression. Current work aims to assess how quickly this compaction happens after formation of a new polyploid, and we also aim to assess different histone modifications that mark active and inactive genes
The data generation is complete: RNAseq; small RNAs; ATAC-seq; HiC proximity ligation coupled to ChIP, and ChIP for active histone marks; bisulphite sequencing for DNA methylation analyses, skim sequencing to assess genome integrity; karyotyping; and RNAseq of deletion lines.
We have concluded the data generation phase of this project and developed a genomics framework for comparing gene expression and chromatin states between different lines. Initial analyses show the rapid imposition of new patterns of dominant and repressed gene expression. Some patterns are conserved between different crosses, which some appear to be specific to each cross, suggesting stochastic events may be occurring. Chromatin accessibility patterns are rapidly altered. Our work is starting to demonstrate a central role for chromatin states in imposing and maintaining new patterns of gene expression in newly formed polyploid lines
Exploitation Route Yes, epigenetic markers could be developed to provide tools for selecting desirable epigenetic states in wheat lines.
Also, our work may provide some predictability for selecting lines for new synthetic wheat production.
Sectors Agriculture, Food and Drink

 
Description We have just started the grant so this question is irrelevant. However, excellent progress is being made, with two consortium meetings setting up a detailed research plan, which is now well underway. The importance and novelty of this work is now very apparent. UPDATE: new hybrids have been made and RNA and DNA extracted from multiple triploid lines. Related lines have been treated to double chromosome numbers and these will soon be sampled. Sequencing of small RNA, ribo-depleted RNA and gene capture bisulphite sequence is ongoing. ATAC sequencing is being optimised in wheat to map any changes in chromatin accessibility upon polyploidisation. We are well advanced into the project and have taken a new approach that explores the role of chromatin in establishing and maintaining new patterns of gene expression in wheat hybrids. This is turning out to be be productive and is providing fundamental new insights into polyploidy We have completed the extensive data-generation phase of this project and established a genome bioinformatics framework for data integration and analyses. The initial results look very exciting and we anticipate a high impact publication to be ready by the end of 2020.
First Year Of Impact 2019
Sector Agriculture, Food and Drink
Impact Types Economic

 
Title ATAC-seq for wheat 
Description ATAC-seq is a sequence- based assay for measuring the accessibility of chromatin to proteins. Chromatin accessibility locates regions of DNA in genomes that can regulate gene expression, and the extent of chromatin accessibility indicates the potential of an adjacent gene for expression. This method was developed for use in human cell lines. We adapted the method for plants- specifically wheat. This involved substantial work on rapid purification of clean nuclei and downstream data processing to remove contaminating reads of organellar origin. This method provided new insights into chromosomes in newly formed polyploid wheat lines. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact We have been able to provide evidence that rapid changes in chromatin states underly changes in gene expression caused by very recent polyploidisation. This is an important finding with respect with new synthetic wheat lines, which often exhibit new and useful traits 
 
Title eHiC "Easy proximity ligation assay" for wheat 
Description Proximity ligation is a powerful method for mapping the relative locations of DNA stands in intact chromatin. Such interactions are increasingly seen as important for understanding the interactions between chromosomes and how these relate to gene expression. There have been several variant methods developed for human cell lines, each with improvements and specialisations. We wished to apply this method to wheat in order to test the hypothesis that newly formed homoeologous genes and regulatory loci have a common regulation due to physical interactions, and to measure any changes in chromosome- scale compaction. In wheat proximity ligation needs to be very efficient due to the large size of the genome. We have adapted a recently published method from human cells that promised to be more efficient in recovering the desired types of ligation products. Our adaptations have substantially improved the recovery of inter-locus ligation events. This means the method can be coupled to chromatin IP to enrich for different chromatin states. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact We are using the new eHiC method on new wheat synthetic lines to assess the role of very recent polyploidisation on chromosome interactions. Specifically we are assaying genes whose expression is dominant or repressed in particular genomes. The outcomes of this work will provide fundamental new insights into the emergence of new traits in polyploid crops such as wheat and into whether and how gene regulatory loci interact 
 
Title Supporting data for "Integrating genomic resources to present full gene and putative promoter capture probe sets for bread wheat" 
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. 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. 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 Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
 
Description Cold Spring Harbor Laboratory 
Organisation Cold Spring Harbor Laboratory (CSHL)
Country United States 
Sector Charity/Non Profit 
PI Contribution CSHL partnership provides access to their sequencing laboratory and expertise in sequencing small RNAs
Collaborator Contribution sequencing RNA and small RNA from new wheat hybrids
Impact RNAseq data sets for analyses
Start Year 2017
 
Description Wheat eipgenomics 
Organisation Cold Spring Harbor Laboratory (CSHL)
Country United States 
Sector Charity/Non Profit 
PI Contribution This project started in July 2016, and aims to study the epigenomes of wheat and progenitor species. Current work involves 1. methyl-sequence of the complete CS42 reference genome. This sequencing has almost been completed at CSHL 2. Create new hybrids of wheat and study how genome interactions influence genome methylation patterns and gene expression 3. Relate methylation patterns to histone methylation patterns and chromatin conformation
Collaborator Contribution 1. CSHL are carrying out bisulphite sequencing, experimental design and data interpretation 2. EI are carrying our exome capture, bisulphite sequencing and chromatin IP 3. NIAB are creating new hybrids
Impact too early for completion of analyses of large datasets
Start Year 2016
 
Description Wheat eipgenomics 
Organisation Earlham Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution This project started in July 2016, and aims to study the epigenomes of wheat and progenitor species. Current work involves 1. methyl-sequence of the complete CS42 reference genome. This sequencing has almost been completed at CSHL 2. Create new hybrids of wheat and study how genome interactions influence genome methylation patterns and gene expression 3. Relate methylation patterns to histone methylation patterns and chromatin conformation
Collaborator Contribution 1. CSHL are carrying out bisulphite sequencing, experimental design and data interpretation 2. EI are carrying our exome capture, bisulphite sequencing and chromatin IP 3. NIAB are creating new hybrids
Impact too early for completion of analyses of large datasets
Start Year 2016
 
Description Wheat eipgenomics 
Organisation National Institute Of Agricultural Botany
Country United Kingdom 
Sector Private 
PI Contribution This project started in July 2016, and aims to study the epigenomes of wheat and progenitor species. Current work involves 1. methyl-sequence of the complete CS42 reference genome. This sequencing has almost been completed at CSHL 2. Create new hybrids of wheat and study how genome interactions influence genome methylation patterns and gene expression 3. Relate methylation patterns to histone methylation patterns and chromatin conformation
Collaborator Contribution 1. CSHL are carrying out bisulphite sequencing, experimental design and data interpretation 2. EI are carrying our exome capture, bisulphite sequencing and chromatin IP 3. NIAB are creating new hybrids
Impact too early for completion of analyses of large datasets
Start Year 2016
 
Description Attendance at ERA-CAPs coordination meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Prof Bevan attended the ERA-CAPs coordination meeting in Lisbon for discussions on how to address major challenges to secure future food supplies and a viable bio-economy in Europe.
Year(s) Of Engagement Activity 2015
 
Description Presentation at PAG conference San Diego USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented our latest data on comparative gene expression between diploid and hexaploid wheat lines
Year(s) Of Engagement Activity 2019
 
Description planning wheat genome analyses 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An annual meeting to plan multiple aspects of wheat genome and epigenome sequencing
Year(s) Of Engagement Activity 2018