Progress 03/01/19 to 02/28/22
Outputs
Target Audience:Primary target audiences include researchers and crop breeders who will be served by availability of epigenome-linked trait variation that will be useful in crop improvement. End-use audience members will include consumers of crops affected by value-added trait variation resulting from identification and deployment of epigenome-based trait improvement. Changes/Problems:As noted in the above reporting sections, we added a project activity in terms of persuing targetted modification of methylation in the promoter of a fruit ripening gene. Said methylation is variant in a line with latered ripening and co-segregates with the delayed ripening phenotype. Working in collaboration with colleagues at UCLA we were able to access constructs they had developed for CRISPR/Cas9-mediated targetting of DNA methylation enzymes to specifically alter the candidate locus. Constructs have been developed and trangenic plants are being generated for subsequent analysis at the writing of this report. This will be a rare and unique opportunity to functionaly address heritable genome methylation differens and their role in conferring a genetically linked ripening phenotype. What opportunities for training and professional development has the project provided?Under this project one postdoctoral scientist, one Ph.D. student and two undergraduate summer interns received training in aspects of analysis including plant propagation, trait phenotyping, carotenoid analysis, development of DNA constructs and targetted gene editing. In short, the postodc was involved in all activities while summer interns focused on plant propagation and phenotyping while the summer interns particpated in carotenoid analyses and also participated in poster development and presentations at a summer student symposium on the Cornell University campus. How have the results been disseminated to communities of interest?Data is still being analyzed and we expect to publish at least two manuscripts on a) phenotyping and genetic analysis of epigenome-realted phenotypes in the Epi-RIL tomato population and b) targetted manipulation of specific epi-alleles for functional confirmation. While project funds have been expended, activiites and our collaboration with our UK partners will continue until completed (apx. 8 - 12 months) - in our case using discretionary USDA-ARS research funds. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The Epi-RIL population was phenotyped under both field and greenhouse conditions during the course of the project with phenotyping for crop production, fruit development and fruit quality traits as noted above. Metabolite data was colected from ripe fruit of our filed trial and from a parallel triual done in the UK by our collaborators. A third trial was performed in the greenhouse for a subset of lines displaying consistent phenotypes in both the greenhouse and field trials. From this third trial lines with consistent phenotypes from all three trials and with the UK field trial were crossed to develop segregating populations that will be phenotyped in an effort to map Epi-RIL loci during summer 2022. A specific epi-allele assocaited with fruit ripening is also being targetted via gene-specific DNA methylation and de-methylation in corresponding methylated and unmethylated genotypes. This is being accomplished via use of the SUN-TAG system to direct DNA methyltransferase and demethylase activities to the target locus. Transgenic plants are being generated at the wrting of this final report and should be available for analysis in 3-6 months to analyze and confirm effects of altered methylation at the suspected epigenome modified locus. This last objective was beyond the original scope of the project but was persued as the opportunity and technology to functionally address a candiate methylation variant was became available.
Publications
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Progress 03/01/20 to 02/28/21
Outputs
Target Audience:Project activities were presented in two seminars presented by the postdoc and Ph.D. student respectively on the Cornell campus. The project PI also presented this project to a groups of 30+ summer interns as an introduction to the importance and potential utility of epigenome analysis and utilization. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoc involved in the project broadened her collaborations to include interactions with experts in DNA methylation who are developing technologies for targeted locus-specific genome modification of DNA methylation.Possibly beyond the scope of the project, but we hope to modify specific loci revealed in further DNA methylation analysis of the tomato epiRILs to validate observed changes associated with candidate genes underlying modified phenotypes. A Ph.D. student working on fruit folate synthesis and accumulation participated in this reporting period to bring analysis of this important nutritional trait into the project. How have the results been disseminated to communities of interest?The first two years of the project was mainly focused on characterization of the epiRIL population. As this has been largely completed and activities move to epigenome characterization the research will be better positioned for inclusion in presentations and aspects will begin to be developed for publication. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will focus on whole genome bisulfite sequencing to assess genome wide cytosine methylation profiles in the leaves and fruit of the sub-population with consistent phenotypic variation over multiple trials. These lines are believed to most likely contain heritable epigenome variation influencing said traits.
Impacts
What was accomplished under these goals?
Genetic diversity is the basis of crop breeding and improvement where breeders cross genetically different individuals of the species to generate novel varieties with desirable combinations of genetic traits. The ability to bring further improvements, for example in crop yield, pest resistance, climate resilience, texture, flavor or nutritional quality, is dependent on available genetic diversity within a species. The genetic information of an organism is captured in its total DNA sequence or genome and through additional heritable chemical modifications of DNA. These modifications contribute to the epigenome and one such chemical modification is DNA methylation. Epigenome diversity has been largely overlooked to date as a means toward crop improvement. This project is characterizing a population of tomato plants harboring essentially identical genomes but with plant-to-plant variation in DNA methylation. The overall project objective is to assess the potential for use of epigenome variation as a means of broadening the genetic tools available for breeders to improve crops. During the current project period a total of 96 tomato epiRIL lines were grown in a field plot in Freeville, NY. Five to six replicate individuals were grown per line in a randomized trial. All 96 lines were grown and phenotyped in the last reporting period in a greenhouse trial which additionally included 8 accessions that did not germinate in sufficient numbers for the field trial. Measured filed phenotypes included those as in the prior greenhouse trial (yield, plant dry matter, plant height, time to flowering, time to initial ripening and full ripe, fruit size, leaf and fruit chlorophyll, fruit carotenoids and an untargeted metabolite analysis of polar compounds). We also included measurement of ripe fruit ethylene and folic acid. Stored tissues from the prior greenhouse trial were also assayed for folic acid. Combined data analysis with that from the prior greenhouse experiment and data from our UK collaborators (who also performed a greenhouse trial of this population) was used to identify 12 lines meeting the criteria of 1) consistent deviation from parental controls in all three trials and for multiple phenotypes, 2) multiple phenotypes with variation in the top or bottom 20% of extremes of the population, 3) lines with no other gross abnormalities of plant growth or form. This latter point is relevant as several lines that displayed the most phenotypic variation in all traits measured were clearly very different than most accessions, presumably due to severe defects in normal developmental process that influenced virtually all other traits. In short, this sub-population was selected as most likely to reveal measurable differences in DNA methylation associated with changes in specific traits. This sub-population was again grown in the greenhouse to validate phenotypes and while all showed similar changes as in prior trials, 8 that displayed one or more fruit phenotypes were selected for further analysis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Fenn M, Giovannoni, J. (2021) Phytohormones in Fruit Development and Maturation. Plant J. 105: 446-458
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:IN the initial phase of the project the primary audicne was lab members, collaborators and USDA-ARS, Boyce Thompston Institute and Cornell University students and faculty who participated in local research presentation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoc involved in the project has her Ph.D. in plant physiology and has received training in genetics and epigenetics thus broadening her portfolio of research skills. She has also had opportunities to interact with international collaborators specializing in metabolite analyses. the undergraduate involved in the project had no prior laboratory skills and worked with the postdocduring the summer gaining experience in plant growth, phenotyping, tissue preparation and cryogenic storage in addition to general lab safety and routine molecular techniques. How have the results been disseminated to communities of interest?As the project was initiated in this reporting period the only dissemination of results has been to project collaborators and through local presentations to students and faculty. What do you plan to do during the next reporting period to accomplish the goals?As the project was initiated in this reporting period the only dissemination of results has been to project collaborators and through local presentations to students and facultAgreenhouse trail of the epiRILs and associated phenotyping was performed in year 1 and compared to results from a greenhouse trial performed in the UK. In the next reporting period we will focus mainly on a field trial of the same population to be followed by selection of genotypes with consistent phenotypic variation in a range of monitored phenotypes across all trials. These are most likely to reflect heritable epigenome variation influencing said traits. We will then focus on individual genotypes for genome, epigenome and transcriptome analyses to better understand the changes which may underlie DNA methylation-based heritable trait variation.
Impacts
What was accomplished under these goals?
Genetic diversity is the basis of crop breeding and improvement where breeders cross genetically different individuals of the species to generate novel varieties with desirable combinations of genetic traits. The ability to bring further improvements, for example in crop yield, pest resistance, climate resilience, texture, flavor or nutritional quality, is dependent on available genetic diversity within a species. The genetic information of an organism is captured in its total DNA sequence or genome and through additional heritable chemical modifications of DNA. These modifications contribute to the epigenome and one such chemical modification is DNA methylation. Epigenome diversity has been largely overlooked to date as a means toward crop improvement. This project is characterizing a population of tomato plants harboring essentially identical genomes but with plant-to-plant variation in DNA methylation. The overall project objective is to assess the potential for use of epigenome variation as a means of broadening the genetic tools available for breeders to improve crops. The project was initiated during this reporting period. A population of 115 tomato epiRILs was secured from our collaborators at INRA in Bordeaux, France. This population was derived from an initial cross between a transgenic line repressing a DNA methyltransferase (MET1) via RNAi and a non-transgenic line of the same genotype. The lines were developed by selfing F2 individuals which had segregated out the transgene and thus should be largely fixed for any heritable modifications in DNA methylation. During this reporting period all lines were grown in triplicate in a greenhouse and phenotyped for plant height, flowering time, time to initial fruit ripening, time to full ripe, fruit yield, fruit size, fruit and leaf chlorophyll content, fruit carotenoids and whole plant dry weight following fruit removal. Ripe fruit were additionally subject to an untargeted analysis of polar metabolites yielding data on 76 known compounds including sugars and organic acids. All phenotypes exhibited variation across the population and high and low extremes were noted and compared to a similar trial run by our collaborators in the UK. Leaf and ripe fruit tissues were cryogenically stored for future analysis and RNA was extracted and stored from ripe fruit of all lines and parents. At the end of the project period seed was allocated in preparation for a field trial to be initiated in the spring of 2020. While the COVID-19 pandemic started during this reporting period the nature of activities performed (much greenhouse work facilitating separation and willingness of the postdoc to work off hours in the lab) allowed completion without interruption.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Fraser P, Aharoni A, Hall R, Huang S, Giovannoni J, Sonnewald U, Fernie A. (2020) Metabolomics should be deployed in identification and characterization of gene-edited crops. Plant J. 102:897-902
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