Robert A Martienssen - US grants

Nuclear genes are likely to be responsible, in large part,for the coordination of chloroplast biogenesis and leafdevelopment in higher plants. Nuclear mutations can identifysuch genes even when they do not encode abundant chloroplastproteins, and have been widely used in the analysis ofchloroplast development. However, pleiotropic effects,particularly in the case of pigment deficient mutants, tendto obscure the biochemical lesion responsible for most mutantphenotypes. Transposon mutagenesis and cloning provide analternative approach to the analysis of these mutations. Thesubject of this proposal is the non.photosynthetic maizemutant hcf*106. This mutation has a dramatic effect onthylakoid membrane organisation a key, morphological processduring chloroplast development. Hcf*106 membranes are notdeficient in pigment or lipid biosynthesis, but fail toaccumulate a subset of thylakoid proteins. The mutant genehas been cloned using the transposon Mul as a molecular"tag" and genetic arguments are presented demonstrating theidentity of the clone. The experiments outlined in this proposal are designed todetermine the molecular function of the hcf*106 gene productin thylakoid membrane elaboration, and should provideimportant insights into genetic regulation of chloroplastdevelopment.

This award provides aid in the construction of plant growth facilities to be shared by a group of investigators who do biochemical and genetic research on corn and Arabidopsis. Growth facilities that provide reproducible conditions for plants are crucial to the kinds of careful, quantitative analysis needed for the effective use of plants in modern biology. Moreover, such facilities increase the productivity of plant researchers since the growing season for plants like maize can be extended to cover the fall and winter months. The award will augment the capacity for growth of corn at the Cold Spring Harbor Laboratory by 50% and will provide for an even greater augmentation for growth of Arabidopsis, a plant particularly well suited to basic studies of plant growth, development and reproduction.

The maize mutant iojap has been a model system in the study of nuclear-plastid interactions in higher plants since the work of Jenkins (1924) and of Rhoades (1943). Plants homozygous for this nuclear mutation have variegated leaves, and transmit defective chloroplasts through their female gametes. Maternally inherited defective plastids have suffered an irreversible change that cannot be rescued by the nuclear genotype of the zygote, resulting in albino seedlings whose plastids are incapable of differentiation into chloroplasts. Furthermore, the pattern of leaf variegation observed in homozygous individuals suggests that the gene product is a key component of the regulatory machinery that co-ordinates cellular and plastid development in higher plants. We have recently obtained a molecular clone of the iojap locus by transposon-tagging using the maize transposon Mu1. In this proposal we describe experiments intended to determine the role of the Iojap gene product in nuclear-plastid interaction and the basis for the maternal inheritance of the defective plastids. We shall also initiate genetic studies aimed at determining the developmental parameters involved in the patterns of leaf variegation observed in mutant plants. The proposed study will contribute to our understanding of the mechanism by which nuclear-encoded proteins regulate chloroplast biogenesis during leaf development in higher plants. It might also provide a molecular explanation for the patterns of leaf variegation and maternal inheritance observed in this and other higher plant mutants of this type. We hope to distinguish between a number of genetic mechanisms that have been postulated to account for these patterns, and so further our understanding of pattern formation in plants and other multicellular organisms.

High chlorophyll flourescent (hcf) mutants of higher plants represent a class of nuclear genes required for the development of photosynthetically active chloroplasts. Their products may include nuclear-encoded components of the photosynthetic apparatus, as well as enzymes and regulatory genes required for co-factor biosynthesis and plastid gene expression. Some hcf mutants have pleiotropic effects on membrane protein assembly and thylakoid organization. hcf106 is an example of such a mutation in maize: The Hcf106 gene has been cloned by transposon tagging, and the gene product shown to be an integral chloroplast membrane protein. It has a single transmembrane domain, a potential nucleotide binding site, but no other homology to known proteins. Thylakoid membranes from mutant chloroplasts have lost their characteristic lateral heterogeneity, and fail to accumulate a subset of thylakoid membrane proteins. Analysis of derivative alleles, hypoploids and double mutants will be used to genetically define the hcf106 lesion. Immunolocalization by suborganellar fractionation and immuno- electron microscopy will be used to investigate the possible roles of this protein in thylakoid assembly. %%% Photosynthesis is the basis of life on the planet Earth and a complete understanding of the process as well as chloroplast biogenesis is one of the most important goals of biological research.

Abstract 9408042 An in vivo gene-trapping technique has been developed in Arabidopsis using the transposable elements Ac and Ds. Our modified Ds element (DsG) contains a Beta-glucuronidase reporter gene that is equipped with splice acceptor signals to allow multi- frame gene fusions (an "exon-trap"). When this Ds element inserts into a chromosomal gene, the pattern of chromosomal gene expression is mimiced by the Beta-glucuronidase reporter gene. Because expression depends on gene fusion, insertions that result in reporter gene expression will also result in gene disruption. Using a novel selection scheme, a collection of 5,000 plants will be generated that will each carry an insertion at a different location in the genome. Insertions into genes will be identified by staining the progeny of each plant for reporter gene expression: 15-20% of DsG insertions result in reporter gene expression, so that the proposed collection should include insertions into 500- 1,000 genes. We will use this collection to develop an exon-trap sequencing technique to enable the rapid sequencing of a portion of the chromosomal gene identified by each exon-trap tag. The chromosomal exon corresponding to each insertion will be amplified by RACE PCR, and its sequence determined. As many of the exons as possible will be mapped via anchored YAC contigs and recombinant inbred lines. A database of DsG insertion lines will be developed, each characterized by expression pattern, mutant phenotype and map location, as well as partial exon sequence. This database can be integrated with existing genomic and cDNA sequence databases. *** A major goal in the characterization of the Arabidopsis genome is to assign a function to genes identified by cDNA and genomic sequencing, gene-finding algorithms, and map location. Homology searches are expected to fulfill this goal for those genes that have similarity to sequences from other organisms. However, a majority of gene products will remain an onymous in these large collections of sequenced genes. In the proposed study, a function will be assigned to many of these genes by determining their patterns of expression, and by observing the mutant phenotype that results from gene disruption. This proposal will form the basis for establishing a large database of insertion lines, as other laboratories use the "starter" lines to generate collections of their own. This will allow a function to be assigned to many of the genes identified by DNA sequence alone, as well as providing a genetic resource of widespread use. %%%

9723671 Martienssen In a few years, the DNA sequence of the Arabidopsis genome will be known. However, there will be many coding regions for which we do not know a function and the developmental context and location in which they are expressed will not be known. The goal of the project is to generate new lines of Arabidopsis each carrying a gene trap or enhancer trap transposon and to sequence the genomic DNA flanking a number of the trapped insertion sites. This work will form the basis for a larger database which can eventually be merged with the genomic sequences, to provide data on cell-specificity and developmental specificity of unknown genes. The collection will become a powerful resource for fine-mapping and positional cloning as well as for local saturation mutagenesis.

The genetic makeup of crop plants is the fundamental basis for yield, grain quality, plant breeding and crop improvement. In principle, this genetic makeup can be determined by sequencing plant DNA, and in this way identifying most if not all of the genes encoded by a given plant genome. Sophisticated software tools are becoming available that allow a great deal of information to be extracted from sequence data, but ultimately the function of a gene can only be determined by genetic analysis. Classically, this has been achieved by subjecting plants to mutagenizing agents, such as chemicals or X-rays, and then searching for those mutants that have a desired property or trait. The gene mutated in each case can then be identified by a laborious genetic procedure to ultimately isolate the corresponding sequence of DNA. The availability of large amounts of methodically determined DNA sequence information, however, allows a novel, systematic approach to be taken to determine gene function. This approach, first pioneered in model animal genomes such as the fruitfly Drosophila and the nematode worm, C. elegans, involves first constructing a library of thousands of organisms, each of which has a different spectrum of mutations. This library is then screened directly with a DNA sequence to identify mutations in a given gene. Two technological advances make this possible. The first is the use of transposable elements as the mutational agent. Transposable elements were first discovered in maize at Cold Spring Harbor Laboratory by Dr. Barbara McClintock in the 1940s. These elements have a conserved DNA structure enabling them to be readily identified in the genome. They disrupt genes at random, simultaneously providing the mutation of interest, and labeling the gene with the conserved DNA sequence. The second technology is the polymerase chain reaction (PCR), which allows such DNA sequences to be amplified from individual plants giving immediate access to a plant carrying a given mutation. Scientists at Cold Spring Harbor Laboratory have combined these advances to develop a systematic method for determining gene function in maize. Robertson's Mutator transposable elements, first characterized at the DNA level at the University of California at Berkeley, are uniquely suited for this purpose, and a sophisticated genetic strategy has been developed allowing large populations of plants to be screened for mutations in pools. In partnership with Novartis AG, geneticists at Cold Spring Harbor and Berkeley are developing a population of 40,000 plants, and DNA samples are being extracted. Seed from each plant will be cataloged and stored. The PCR reaction will be performed on DNA pools derived from this population so that seed corresponding to mutations in a given gene can be readily identified. Individual geneticists from the maize community will be able to send in sequence information for targeted disruption of genes of interest to them. The information will be entered into a database, and seed corresponding to the mutation will be distributed to interested researchers to allow them to determine the function of each gene chosen in this way. The Mutator Targeted Mutagenesis (MTM) system can thus be used to build up a database of gene function in maize, eventually comprising a significant proportion of the genetic makeup of this crucial crop plant.

This award supports the acquisition of a scanning electron microscope (SEM) that operates under conventional high vacuum as well as variable pressure mode. The instrument will by used by at least seven research groups at the Cold Spring Harbor Laboratory to study the developmental genetics of plants and animals. Projects that will use the microscope include the analysis of leaf and meristem development in maize and Arabidopsis; analysis of the Arabidopsis flower and ovule development; the developmental genetics of cell death in the nematode C. elegans; developmental biology of GTPase signaling in Drosophila; and the analysis of morphological changes associated with the development of learning and memory in the Drosophila brain. The SEM is essential for these studies.
The Plant Group at Cold Spring Harbor Laboratory is engaged in a systematic search for new mutations in Arabidopsis using gene trap transposon mutagenesis. The SEM will provide a means to rapidly characterize these mutations in terms of morphological changes as well as changes in cell shape or specification. In addition the SEM will be used for training graduate students and postdoctoral fellows, and in the Cold Spring Harbor Laboratory course in Arabidopsis molecular genetics, which has a wide impact on the plant field, and in the DNA Learning Center at CSHL. The ease of use of the variable pressure SEM, compared to conventional SEMs, allows samples to be visualized within minutes of dissection without the need for extensive sample preparation, and the automated settings and the standard operations in 'Microsoft Windows' format means that users can be quickly trained. Therefore the impact of this microscope on research and education at Cold Spring Harbor Laboratory will be substantial.

The maize genome probably contains between 50,000 and 100,000 genes clustered in hypomethylated gene rich regions. We have developed a new method for purifying these regions, and we will explore its use in identifying gene function. This will be accomplished by sampling gene-rich portions of the genome that are chemically distinct from the gene poor regions, and then displaying them on microarrays (DNA chips). These microarrays will then be queried using representations of the DNA corresponding to mutated genes from a large population of maize plants that carries transposable elements (jumping genes). We have previously developed one such population (the Maize Targeted Mutagenesis population) with prior funding from the Plant Genome Research Program. By using computational methods, we will combine the data from the two different resources to create an index of maize gene function, represented by maize seed corresponding to each mutated gene. We anticipate making thousands of entries into this index, allowing maize geneticists to systematically determine the functions of indexed genes.

DESCRIPTION (provided by applicant): This application requests support for the Fifth Gordon Research Conference on Epigenetic Regulation of Gene Expression to be held at Holderness School, New Hampshire, August 10-15, 2003. Epigenetic regulation is mediated by the creation and maintenance of heritable, but potentially reversible, changes in chromatin structure and/or DNA methylation, which alters gene expression without altering DNA sequence. Epigenetic effects have been discovered in many organisms and they comprise some of the most intriguing and actively investigated phenomena with relevance to both basic and applied science. For example, loss of epigenetically imprinted events on the chromosome contribute to tumor progression in many human cancer types. In addition, mutations in genes encoding DNA methyltransferase and a methyl-DNA binding protein lead to mental retardation in the form of immunodeficiency, centromeric instability, and facial anomalies (ICF syndrome) and Rett syndrome, respectively, while loss of imprinting leads to childhood diseases such as Angelman and Prader-Willi syndromes. There is increasing evidence that epigenetic silencing contributes to variation among clones, which has profound implications for stem cell research in the biomedical arena, as well as for the cloning of farm animals in agriculture. Remarkably, the molecular mechanisms that underlie epigenetic silencing are conserved in fungi and in plants, where they impact transgene silencing and somaclonal variation, as well as contributing significantly to natural variation and a host of classical genetic anomalies, such as paramutation. There has been tremendous progress in elucidating these mechanisms in the last few years and this will be a major focus of this conference. Invited speakers are leading researchers working on fungal, plant, and animal models as well as human disease, who will cover topics such as control and function of DNA methylation, histone modifications, RNA interference, imprinting, X-inactivation, prions, epigenetics and disease, genome defense systems, paramutation, and position effects. The Epigenetics Gordon Conference provides a unique opportunity for researchers working on related phenomena in different organisms to come together and exchange recent results and ideas. It is in a cross-disciplinary environment such as this that intellectual leaps occur and innovative ideas flourish.

? DESCRIPTION (provided by applicant): The germlines of plants and animals undergo genome reprogramming in order to reset epigenetic marks that would otherwise interfere with pluripotency of the zygote. Perhaps chief among these marks are epigenetic modifications of transposable elements (TE), which make up a majority of most eukaryotic genomes. We have found that small interfering RNA derived from heterochromatin plays a key role in germline reprogramming in plants, and there is mounting evidence for a similar phenomenon in animals. Germ cells in plants differentiate from the products of meiosis by mitotic division, along with companion cells that resemble nurse cells and other support cells in animals. We have found that reprogramming of the pollen grain companion cell nucleus (the vegetative nucleus or VN) results in transposon activation, and that a new class of epigenetically activated small interfering RNA (easiRNA) accumulate in sperm cells, that have the potential to silence these same transposons. We have recently found that genome reprogramming depends on DNA methylation, but also on the deposition and modification of specific histone variants, that is likey the result of cell cycle dependent chromatin remodeling. Epigenetic inheritance is far more widespread in plants than in mammals, although the mechanisms are largely conserved, so that plants provide an excellent system to study their origin. We will investigate the mechanism of germline reprogramming in plants, and the transgenerational consequences when reprogramming goes awry.

PI: Gregory J. Hannon (Cold Spring Harbor Laboratories)
CoPIs: Robert A. Martienssen and W. Richard McCombie (Cold Spring Harbor Laboratories)

Determining the function of all the genes in the model plant, Arabidopsis thaliana, will have a profound impact on both our understanding of plant biology and our ability to enhance desirable traits in agriculturally important plant species. Conventional methods for determining gene function generally rely on creating mutant versions of genes that are non-functional. This is accomplished by altering the gene itself and subsequently bringing both copies of a non-functional gene together in one plant. This strategy works for most of the ~30,000 Arabidopsis genes, which are present in the genome at single copy. However, roughly 8,000 Arabidopsis genes exist as families of closely related copies that are much more difficult to coordinately inactivate by conventional methods. The goal of this CSHL 2010 project is to develop resources that allow the function of the genes that form these multi-gene families to be determined. To accomplish this, the project will take advantage of a conserved biological pathway that acts as a programmable engine for gene silencing. This pathway is known as RNA interference or RNAi. In response to double-stranded RNA, RNAi turns off the expression of any gene that shares sequence with the silencing trigger. Unlike conventional mutants, one copy of an artificial gene expressing double-stranded RNA is sufficient to silence all members of even a multi-gene family. Moreover, double-stranded RNA production can be regulated in a manner that restricts the inactivation of genes to specific tissues or times in development. RNAi triggers against each of the 8000 genes that are present as members of Arabidopsis multi-gene families will be produced. Additionally, RNAi triggers against genes that are essential early in development will be created, as the ability to turn off the activity of these genes in selected tissues or in a timed manner may reveal aspects of their function that cannot be illuminated by conventional mutations. Resources developed through this project will complement existing mutant collections, as conventional tools are not suited to determining the function of genes within multi-gene families and genes with early lethal phenotypes. Without such complementary approaches, the goal of understanding the function of all genes in Arabidopsis by 2010 will be unattainable.

All resources will be made readily available to the scientific community as they are developed through a public repository, the Arabidopsis Biological Resource Center and through a commercial reagent distribution network, Open Biosystems. Access to information regarding the status of the resource, the overall project and how to obtain materials can be obtained from the project website at http://2010.cshl.edu. Information about the participating investigators and their research programs are also available. With respect to outreach and training, the project will leverage efforts at Cold Spring Harbor Laboratories through the Dolan DNA Learning Center to incorporate the plant resources generated into their curriculum.

DESCRIPTION (provided by applicant): Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. It is inherited through mitosis and has roles in transcriptional silencing, centromere specification and genome integrity, which profoundly impact epigenetic mechanisms in human health and disease. We have found that the epigenetic inheritance of heterochromatin in fission yeast requires RNA interference (RNAi) to guide histone modification, which occurs during the DNA replication phase of the cell cycle. In the fission yeast S.pombe centromeric repeats have an alternating arrangement of small RNA clusters and origins of replication that makes collision of the transcription and replication machineries all but inevitable. We propose that RNA interference promotes release of RNA polymerase (PolII) during S phase, allowing completion of centromeric DNA replication by the leading strand DNA polymerase. DNA Polymerase epsilon directly recruits the histone-modifying Rik1 complex and so can spread heterochromatin along with DNA replication. In the absence of RNAi, stalled forks are repaired by homologous recombination (HR) without histone modification, so that HR is essential in the absence of RNAi. This model may explain the participation of non-coding RNA and DNA replication in many examples of epigenetic silencing, including paramutation in plants, and imprinting and X-inactivation in mammals. S.pombe is an outstanding model system for cell cycle research, heterochromatic silencing, and RNAi. We will examine the roles of DNA replication, RNA Polymerase release, DNA recombination and repair in heterochromatic histone modification mediated by RNAi. We will utilize models of heterochromatic nucleation and RNAi, as well as chromosome profiling and genetic analysis, to test our hypothesis. We will build on our recent results concerning the roles of the Rik1 complex and Centromere-binding protein B in DNA replication and repair, as well as RNA interference.

Epigenetics (inherited changes in phenotype not due to changes in DNA sequence) is a critical mode of gene control recently realized to have far reaching effects. It is caused by modification of the chromatin (DNA plus associated proteins) and acts at a level above normal gene control. How changes in chromatin cause changes in gene expression is a major area of research activity in both animals and plants. Understanding epigenetic control of genes will elucidate gene control of development, response to climatic stresses such as drought and heat and interaction with disease causing organisms. Several plant genomes have now been sequenced so it is feasible to examine the epigenetic changes occurring during development or in response to the environment in these organisms. The goal is to determine, as part of an international collaboration, the epigenetic marks on chromatin at particular genes in different tissues, at different stages of development and in response to biotic and abiotic challenges and to see how they differ, potentially leading to ways of manipulating the expression of these genes. This workshop, to be jointly sponsored by NSF and science funding agencies in the UK and Australia, will bring together approximately 40 scientists, both leading experts and early career, to participate in a forum for discussion of cutting edge research in this area and the setting up of an international Plant Epigenome Consortium. The 'state-of-play' internationally will be reviewed and then the 'nuts and bolts' of setting up a collaboration will be worked out. In addition, early career stage invitees will have the opportunity to visit nniversities and institutes in Australia after the meeting to further these international collaborative goals.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Funding from the MRI program has created a "next generation" sequencing center, dedicated to plant genomics. Such centers were recently called for by a report on the plant genome project by the National Research Council. The facility will be used for a wide range of projects ranging from de novo sequencing of the various plant genomes and transcriptomes to studies of epigenetics. The center will consist of an Illumina GAIIx DNA sequencer and computer equipment for data analysis and storage. Such centers exist of course for human and animal sequencing, but dedicated instruments for plant sequencing will be an important development that would greatly impact plant genomics. The availability of substantial capability in next generation sequencing will allow the much more rapid collection of plant genomics data. The center greatly enhances the national infrastructure for next generation sequencing that is available to the plant genome community, and contributes meaningfully to the training of scientists to use such equipment.

DESCRIPTION (provided by applicant): Cancer research relies on technologies that can interrogate the entire genome, detecting deletions, copy number variants and point mutations responsible for disease. It is becoming increasingly clear that epigenetic lesions in DNA and histone modification are at least as important, and probably more important, than genetic lesions as cells progress towards malignancy, and acquire various attributes, such as drug-resistance. Cancer research at Cold Spring Harbor Laboratory has been at the forefront of this "cancer genomics" approach, as well as research in epigenetic mechanisms that contribute to these and other diseases. Examples of research topics are summarized in the application, ranging from the role of methylation in breast cancer and chemoresistant AML to replication-induced senescence. Further, we plan to examine epigenomic profiles in model animals and plants, which undergo developmentally programmed changes in epigenetic profiles, and which use RNA interference to guide DNA methylation and histone modification patterns. To do this, we are requesting funds to purchase a Genome Sequencer FLX instrument from Roche Applied Science, which can reliably determine the sequence of the epigenome, even when cytosines are converted to uracil, because longer read lengths permit unambiguous mapping back to the reference genome. PUBLIC HEALTH RELEVANCE: As we learn more about how information is stored in our genome and controls biological processes it has become apparent that in addition to our basic DNA structure, modifications to it, such as the addition of methyl groups in specific places is of crucial importance. Changes in methylated DNA regions can change gene expression in normal development as well as lead to cancer if and possibly other diseases if not properly controlled. We are requesting equipment that will allow us to examine, in great detail and at very large scale, the changes to methylation in the genome so that we can better understand how this process occurs in normal biological processes as well as which genes it regulates in cancer.

Abstract

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Funds are provided for Cold Spring Harbor Laboratory (CSHL) to renovate and upgrade existing plant growth facilities at our Uplands Farm Research Field Station. CSHL has an extensive greenhouse complex offsite where seminal work in Arabidopsis and crop plants has been conducted. The research focus on crop yield and adaptation of crops to substandard conditions has global impact and this will increase with these improved facilities. The greenhouses and growth rooms are used to support the research programs and also support the plant genetics teaching programs of the CSHL Dolan DNA Learning Center. These aging and outdated facilities are inadequate to meet the demands of current genome driven plant biology research. The infrastructural improvements will provide appropriate growing conditions for a greater diversity of plant species and will increase the energy efficiency of the facilities. In the proposed renovations, CSHL intends to:
1 Replace boilers in two smaller greenhouses, and two boiler burner units in the largest of the three greenhouses.
2 Install updated evaporative cooling units in each of the three greenhouses.
3 Install automated ventilation, sunshade, and irrigation systems in all three greenhouses.
4 Replace the aging, hazed acrylic sheathing on the largest greenhouse.
5 Renovate and improve the head house of the largest greenhouse.
6 Replace the cooling unit in the Field Station Laboratory with a modern and more efficient unit with an economizer ventilation unit.
7 Replace outdated lighting fixtures in the Arabidopsis growth facilities with improved, high-efficiency units and install a dedicated heating loop.

PI: W. Richard McCombie (Cold Spring Harbor Laboratory)

Co PI: Doreen Ware (Cold Spring Harbor Laboratory)

Key Collaborator: Robert A. Martienssen (Cold Spring Harbor Laboratory)

Genomic sequence data holds the promise of dramatically advancing both the understanding of basic plant science, and of catalyzing practical advances in plant breeding. While the "complete", contiguous, "base perfect" sequence of a genome is the most useful outcome of a genome project, it is not necessary for most applications. Furthermore, base perfect sequence is prohibitive in terms of both time and cost for very large genomes or large numbers of moderately sized genomes. However, recent technological advances and soon-to-be broadly available technologies provide an attractive option for plant genomics. This project seeks to use next generation sequence technologies to identify the vast majority of genes in three closely related progenitor genomes of wheat (AA, AABB, and DD genome containing species) and hexaploid wheat (Chinese spring). Each genome will be sequenced to about 50x coverage with Illumina sequencers (paired end 100 base reads) and to about 8x-10x coverage using the Pacific Bio-Sciences sequencing technology. In addition, the project will sequence each genome with long range "strobe" reads on the Pacific Biosciences instrument that will amount to about 2 terabases of raw sequence. The sequences will be assembled and annotated for maximum use by the broader scientific community. All raw data, assemblies, and annotations will be released to the public as soon as they are generated and quality checked and prior to any publication that might be pursued.

The broader impact of this project is to solve the data availability problem by providing about 2 terabases of raw genome sequence data to the broader cereal research community as well as the assemblies and annotation of the vast majority of genes in a large number of cereal genomes. It is expected that access to these data will radically accelerate functional genomics in wheat. All data generated in this project can be accessed through the project website, GenBank, and through the iPlant Collaborative (http://www.iplantcollaborative.org/). In addition, project updates will be coordinated with the International Wheat Genome Sequencing Consortium (IWGSC) and made available at (http://www.wheatgenome.org/Projects) and at GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml).

PI: W. Richard McCombie (Cold Spring Harbor Laboratory)

CoPI: Robert Martienssen (Cold Spring Harbor Laboratory)

ERA-CAPS Collaborators: Anthony Hall (University of Liverpool, Liverpool, United Kingdom), Klaus Mayer (Helmholtz-Zentrum Muenchen, Munich, Germany), and Michael Bevan (John Innes Centre, Norwich, United Kingdom)

Wheat is one of the major food crops in the world and specifically in the United States. This project will take a comprehensive approach to define the epigenome of bread wheat, the functional consequences of epigenetic modifications, how the genome is re-shaped, stabilized and inherited in newly formed hybrids, and how the environment may influence patterns of epigenetic modification. These are fundamentally important questions in biology and are necessary for understanding trait variation in wheat hybrids. It is anticipated that this project will have broad impacts across three specific themes. The first of these relates to food security. Food security is an ever-increasing challenge in the face of factors such as urbanization and world population growth. One factor that significantly affects agricultural yields of bread wheat is the epigenetic regulation of high-yielding hybrid lines. A better understanding of this process will help others understand the basic processes involved in the creation and stabilization of hybrid lines and thus improve the efficiency at which they can be derived and contribute to food security. The second broad area where the work will have significant impacts is in the area of climate change. Understanding how changing climate affects epigenetic regulation of an important crop genome will likely have broad implications for the understanding of how agricultural yields will be challenged and can be modified to respond to changing climate. Lastly the project will have broad impact in the area of enhancing scientific education and understanding. Rapidly advancing science and technology presents a challenge in conveying a general understanding of that technology and the science behind it to the general public. This project will be used as a platform to better explain science to the general public using different and new educational tools. In addition to the training of students, the project will provide summer international exchange research training for graduate students associated with the US-EU consortium.

Wheat is a polyploid arising from recent hybridization of goat grass (Aegilops tauschii, DD) with wild tetraploid emmer wheat (Triticum dicoccoides, AABB) to form hexaploid bread wheat (Triticum aestivum AABBDD). This process of hybridization is critical to achieving the high yields produced by modern wheat varieties. However, much is not understood about this process particularly in terms of how epigenetics regulates which parental line is expressed within the agriculturally significant hybrids. This project will generate information about the epigenetic regulation of the bread wheat genome and its role in the stabilization of the component genomes within the hybrids. One aim is to understand how epigenetic variation alters gene expression in hybrid wheat by identifying how epigenetic traits or epigenetic marks are set during hybridization in the formation of new wheat strains and how these are maintained in stable wheat hybrids to allow for high agricultural yields. It is the expectation that this project will provide a much better understanding of the molecular mechanisms related to epigenetics that are involved in the creation and stabilization of high-yielding hybrid wheat strains. All data produced will be freely and continuously shared within the consortium. Specifically, all datasets will be accessible through the iPlant Collaborative and European Bioinformatics Institute (EBI) as well as through publicly available data repositories including GenBank.