Progress 09/01/17 to 08/31/20
Outputs
Target Audience:Plant scientists, plant breeders, seed companies are expected to be interested in this new epigenetic breeding method and computational methods for analyzing DNA methylation Changes/Problems:The major project change was the de-emphasis on transgenic RNAi silencing methods and switching to the use of EMS mutant in the target Msh1 gene. This change was due to customer pressure as well as technical benefits. Transgenic RNAi technolog is leaky, and of uncertain cellular specificity in plant development, particularly for heritable epigenetic traits, while a genetic null mutation provides a strong suppression of gene function in all cells at all times of development. Additionally, while RNAi transgenes can become less effective in later generations, a genetic null mutation is very consistent in every generation. What opportunities for training and professional development has the project provided?Valuable experience with breeding, analyzing, and field testing epigenetic materials has been gained by project participants. Valuable computation methods have been developed and improved for analyzing genomic DNA methylation patternshas been gained by project participants. How have the results been disseminated to communities of interest?Publications, scientific meetings, industry meetings, and the recent scientific publication have all be used. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
I. Field Trials with RNAi silenced rootstocks in 2017 and 2018: We successfully performed field trials in Florida in the spring of 2017, and in California and Pennsylvania in the spring of 2018. These field trials demonstrated two key findings: i) We created an "EpiLine" type of heterosis in hybrid tomatoes for a cross between varieties with a 17.5% yield increase above that of the control cross. ii) For graft derived progeny, the epigenetic effect of increased fruit production and increased fruit yields was observed to persist for four (California) and five (Pennsylvania) generations after the graft stage for self-pollinated 'inbred' lines. This considerably increases the number of generations available for the plant breeder and seed company to produce these improved plant lines as inbreds. However, as most commercial seed sales are of F1 hybrid seeds, our key remaining objective is field testing of F1 hybrids. II. Field Trials of F1 hybrids derived from our EMS derived materials in 2019. We performed F1 crosses with elite commercial lines as female with pollen from our Heinz EMS-derived msh1-/- null mutation. These early mutant lines also carried an estimated 400 other EMS mutations, as we had not backcrossed and segregated out these additional mutations that occur in the high-mutation rate EMS method. Our object was to: 1) Examine the phenotype of the F1 plants in the field that are heterozygous for msh1 (the F1 plants are msh1+/-). Result: We did not see any msh1 phenotypes in the field, which means this direct crossing method (no grafts involved) is one possible way to breed for msh1 improved epigenetics. 2) We measured these F1 hybrid field yields at 3 locations in California, with 3-4 replicates per location. Encouragingly, we observed strong seedling heterosis in the seedling trays prior to transplanting them to the field relative to the control F1 seedlings. The field results were a bit ambiguous, although we think we can explain why. At location 1, we had a 30% yield increase in the F1 epigenetic hybrids relative to F1 controls. But in locations 2 and 3, yields were similar to controls. The epigenetic F1 hybrids produced more fruit at all locations, which is a strong characteristic of msh1 epigenetics. The confounding factor was the seedlings, which are transplanted to the field in tomato production, were held in the trays for 3 extra weeks for locations 2 and 3 due to weather and scheduling issues. This also meant the field growing season was 3 weeks less for these two locations. We believe the high load of about 400 other EMS mutations (from the EMS mutagenesis source line) caused the epigenetic hybrids to require a longer season to reach their full yields, which was achieved for location 1 but not locations 2 and 3. Thus, we are encouraged by these 2019 F1 hybrid results, but require additional field trials with genetically cleaner materials to confirm these preliminary results. This is the goal of our 2021 field trials as described below. In 2019, we also collaborated with a second commercial tomato seed company. We grafted our EMS-derived msh1-/- rootstock to two of their elite germplasms, harvested seeds from the elite scion of the graft, and grew these seeds in their 2019 field to look for improved traits. Our seed company partner breeder was very excited by the phenotypes he observed, with some progeny demonstrating more vigor, and more fruit. But this was an observational test, not a replicated field test. Field trials with hybrids of these materials are planned for 2021. For clarity, we also have additional improved materials to be tested in 2021 as described below. III. Field Trials with our EMS derived materials in 2020. Our 2019 results indicated the estimated 400 other EMS mutations likely precluded further direct F1 hybrid testing until most of these other EMS mutations were removed by backcrossing. However, as all DNA mutations stay in the rootstock in a grafting method, we tested grafting methods in a F1 hybrid format in 2020. This means we first grafted our EMS msh1-/- rootstock to an elite scion and harvested seeds from the scion of the graft. These graft-derived seeds were then crossed to a genetically distinct lineage to create genetic F1 hybrids, where one of the parents contained graft-derived epigenetic changes. We performed a single location 4-replicate test that was conducted by PacificAg Research in Huasna, California. At a vegetative state, the epigenetically enhanced plants showed more vigor and biomass, and were a foot taller than the controls. But the plants did not show the msh1 epigenetic signature effect of more fruit or more yield. We conclude a cross where one of the lineages contains epigenetic changes from graft transmission from a rootstock does not increase yields in F1 hybrids, at least for the genotypes tested. IV. Planned 2021 Field Trials. Our EMS-derived msh1 mutation has been introgressed into our seed company partner elite lines to segregate away the undesired other mutations as well as to move our msh1 mutation into the elite genomes. These improved materials are now ready for 2021 field trials. Our data described above have provided us with insights into three methods for introducing our epigenetics into F1 hybrids that will be tested in 2021. We found above that graft-derived epigenetics improves yields in progeny of a graft, but that these lines did not increase yields when crossed to a second 'normal' plant to make F1 hybrid seed. Our hypothesis is that the epigenetics is 'diluted' by crossing to the 'normal' second parent. Therefore, we will test if our msh1 epigenetics will perform as expected if both parents have graft-derived epigenetics. We have made F1 crosses wherein both parent lineages contain epigenetics from grafts to msh1-/- rootstocks. The "EpiLine" crossing method, where the msh1-/- mutant line is crossed to a genetically identical parent to make an isogenic msh1+/- "EpiLine" with improved epigenetics. This epigenetically improved parent line is then crossed to a genetically different parent to make a true F1 hybrid. A true F1 hybrid cross using one parent with msh1-/- epigenetics. This appeared to work in our 2019 F1 hybrid trial described above, and these 2021 materials have had the secondary mutations removed by backcrossing to our seed company partner elite lines. These improved materials should give a conclusive answer for this method. V. Computationally determine the methylome signatures of candidate epigenetic rootstocks (in collaboration, via a sub-award with the Mackenzie group now at Pennsylvania State University at University Park). The collaboration with Dr. Sally Mackenzie, via a subaward, resulted in excellent improvements in MethylIT software computational methods and resulted in a joint publication in a high-profile journal: MSH1-induced heritable enhanced growth vigor through grafting is associated with the RdDM pathway in plants. Hardik Kundariya, Xiaodong Yang, Kyla Morton, Robersy Sanchez, Michael J. Axtell, Samuel F. Hutton, Michael Fromm & Sally A. Mackenzie. Nature Communications volume 11, Article number: 5343 (2020). Two Epicrop researchers were co-authors: my self and Dr. Kyla Morton. This landmark paper describes mechanisms involved in graft transmission of epigenetics, leading to higher yields in field performance of the graft-derived later generations of tomatoes, and improved computational methods of analyzing methylomes of the epigenetically enhanced lines. The MethylIT software computational work improved methods to identify methylation signatures and associate methylation changes with gene expression changes. The computational work also developed improved methods of identifying epigenetic modulated pathways involved in the improved plant yields. This publication should be recognized as a key breakthrough in this important epigenetic area.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
MSH1-induced heritable enhanced growth vigor through grafting is associated with the RdDM pathway in plants. Hardik Kundariya, Xiaodong Yang, Kyla Morton, Robersy Sanchez, Michael J. Axtell, Samuel F. Hutton, Michael Fromm & Sally A. Mackenzie. Nature Communications volume 11, Article number: 5343 (2020).
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:Our main target audience is our potential customers which are tomato seed companies and their employees engaged in tomato breeding and production. We have established partnerships with two tomato seed companies with collaborative research for the purpose of testing our epigenetic technology in their varieties. We have visit their fields with our epigenetic technology in their crops and all are impressed with how it is performing. Changes/Problems:Year 2 is a transition year to the all-important field results to be measured in a no-cost extension in year 3 (2020). The main driver for the no-cost extension has been the project shift from RNAi silencing of Msh1 (a gene that when suppressed created useful epigenetic changes) to a non-GMO, consumer-accepted, Msh1 mutants made via chemical mutagenesis. Comment: I strongly support transgenic and gene-editing research and the USDA's position on these technologies, but our seed company customers are very sensitive to consumer preferences. For business and technical reasons, we have made the decision to use EMS null mutants in Msh1 in our program for two critical reasons: 1) In tomatoes, our seed company partners insist on this EMS mutant approach. 2) We also have some technical data that mutants are more effective for our epigenetic technology (We use stop codon null mutants which are 'null in all cells all the time' as opposed to RNAi which is always leaky in its knockdown efficiency). Therefore, year 2 has been a transition year to move the EMS msh1 mutant lines into the appropriate genetic and epigenetic states for field trials. It takes a minimum of two generations of the homozygous msh1-/msh1- genetics to create a useful epigenetic change. These epigenetic founder lines then need to be crossed or grafted to produce seeds by February of a growing year to fit into the tomato growing dates for summer field testing. This delay is the reason for a no-cost extension to allow for critical field trials in 2020. What opportunities for training and professional development has the project provided?Individuals at Epicrop have improved their scientific skills in breeding, molecular biology, and of course in the novel aspects of epigenetics in the msh1 system we are working with. At a practical level we have also instituted barcoding of samples, and participants are being trained in how to integrate this into all aspects of the plant handling stages. Participants are also being trained in how to prepare samples for winter and summer field trials and good record keeping related to this. How have the results been disseminated to communities of interest?Target interactions with tomato seed company employees have led to two collaborative projects. What do you plan to do during the next reporting period to accomplish the goals?The current year 2 has mainly focused on preparing the plant materials for field trials in 2020, using a no-cost extension to accomplish this. Current results strongly support epigenetic yield gains in F1 hybrids and seed plantedlines developed from grafted parents (using epigenetic transmission via the graft). The 2020 field trials will test this progress in a meaningful way and if as successful as expect, demonstrate a commercially acceptable method for using our epigenetic technology in commercial breeding/production programs - which is the high level goal of the Project.
Impacts
What was accomplished under these goals?
I. We've made excellent progress on commercial acceptance of our EMS-mutant-based,epigenetic technology by working with two tomato seed companies on testing our epigenetic technology in their fields. This is a critical step in commercial implementation of our technology, as our customers are the seed companies that breed, produce, and sell seeds to growers. Company 1: we are introgressing our EMS msh1 mutation into two of their commercial lines. We have reached backcross 2 stage (F1 -> BC1 -> BC2), and have worked closely with this company to use molecular markers across the genome to make the introgression of the msh1 mutation more efficient, in terms of elite parent genotype conversion. As a quick demonstration of our technology in their fields, we used our msh1-/msh1- epigenetic founder tomato variety as a rootstock that was grafted to scions of their two commercial varieties. Seeds from these grafts were then planted in their field in 2019 for observation by their breeders. Their breeder noted fairly dramatic increases in plant vigor, early fruit development, and more extensive fruit development relative to the parent lines. Their breeders are impressed with the phenotypes they are seeing and will move forward with additional testing of these lines. This demonstration created considerable enthusiasm at this company for the technology we are developing. Technically, this represents the first time we have used our EMS-derived msh1 mutants in a crop to develop and pass epigenetic information via grafting to commercial scions. As the rootstock was not genetically identical to the scion, this further demonstrates the usefulness of a set of a 'universal set of epigenetic rootstocks' as a resource to rapidly improve many different varieties. This leads to faster product development. For the above example, we needed just 4 months to perform the graft and collect the seeds, and then send them for field testing (once the epigenetic source was developed). Graft transmission of our epigenetics is dramatic and fast as it uniquely transfers epigenetic information via small RNAs, without transferring any genetic information. This allows for fast epigenetic 'uploads' into existing commercial genomes without changing any of the DNA sequence of those genomes! This is a very unusual biological method and is very fast commercially relative to the usual introgression of a transgene to get the benefits of a new trait. Company 2: in addition to introgressing our EMS msh1 mutation into four of their varieties, we conducted a F1 hybrid 'demonstration' project in 2019 in their fields. In this demonstration, we first made F1 hybrid seeds from a cross of our epigenetic msh1-/ msh1- processing tomato parent with one of their current commercial processing tomato parent lines. These 'epigenetically improved' F1 hybrid seeds were then compared to the control F1 seeds made from a cross the same parent lines, but lacking our epigenetics (no msh1 mutation in their development). Impressively, the 'epigenetically improved' F1 hybrid seedlings were much larger and faster growing than the controls, thereby demonstrating epigenetically-derived heterosis in a commercial variety at the seedling stage. These improved growth rates have persisted after these seedlings were transferred to the field, and the yields from these plants will be measured in September of 2019. As an aside, it is normally quite difficult for a breeder to independently change epigenetics or genetics, making it difficult to assign roles to either - hence the lack of scientific progress on understanding the mechanistic basis of heterosis in plants. Our novel technology allows us to keep the genetics unchanged and change only the epigenetics. This makes it straightforward to measure the impact of our epigenetic changes relative to the control (same genetic parents but lacking the epigenetic changes). In that regard we have a new measurement method for epigenetically created heterosis: a control cross vs a cross of the same genetic varieties with our epigenetics added, where the genetics of both control and treatment are the same. Our result of a F1 hybrid cross of our epigenetic parent with a second genetically different parent, to produce higher yielding F1 hybrid progeny is our first commercial platform. Meaning we have all the steps (msh1 mutation, breeding scheme, and seed production methods) worked out to implement this technology commercially. To summarize the above and what else needs to be done: we have partnerships with two commercial seed companies to test our epigenetic technology. With company 1, we demonstrated increased plant performance in their field using seeds derived from grafted plants (using an epigenetically improved rootstock). We still need to demonstrate increased yields in F1 hybrids using these lines, i.e., this epigenetic graft-derived line will be crossed to a second commercial line to produce F1 hybrid seeds for field testing. This is one of the goals of the no-cost extension field trials in 2020 (with Epicrop conducting the parallel tests with Epicrop materials). With company 2, we demonstrated epigenetically created heterosis F1 in seedlings and plants in the field, with field yield measurements to be made in September, 2019. Importantly, this F1 hybrid method represents a commercially feasible method to bring our technology to market, which is the key goal of this Phase II project. A more complete field test of this result in field trials in 2020 is a second key goal of the no-cost extension (again with Epicrop conducting the parallel tests with Epicrop materials).. II. Summary for: Objective 4. Computationally determine the methylome signatures of candidate epigenetic rootstocks (in collaboration, via a sub award, with the Mackenzie group now at Pennsylvania State University at University Park). As noted in the report for Year1, excellent progress on a novel DNA methylation analysis computational program (named MethylIT) has been made by the Mackenzie group and this program is now available to us in house. In Year 2, we have been successfully using the MethylIT program at Epicrop to identify DNA methylation signatures in our epigenetically modified tomatoes. We have successfully identified the DNA methylation differences between wild type, two independent RNAi lines, and two independent mutant msh1- lines using the MethylIT program on their whole genome methylome data (whole genome bisulfite sequencing of their genomes). The next step is to correlate DNA methylation signatures in different epigenetic source lines with useful epigenetic outcomes (more vigorous progeny that produce higher yielding plants in field trials) in order to find the methylation signature most predictive of desired yield increases. The 2020 field yield results will establish which epigenetic starting materials gave higher yields. By grouping the high and low yielding outcomes, we will be able to identify which DNA methylation signatures in the epigenetic parents are associated with useful field outcomes. Conclusion: MethylIT has been developed by the Mackenzie group to the point where it can successfully identify DNA methylation differences between lines at high sensitivity. This meets Part one of Objective 4's goal of where we have identified candidate methylation signatures in tomatoes with the new MethylIT computational program. MethylIT software is a major milestone in improved sensitivity and methods for detecting DNA methylation patters for the scientific community relative to current methods. Correlation of the DNA methylation signatures with high and low yielding lines derived from our epigenetic ancestor plants is the last goal of our no-cost extension.
Publications
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:Target audience: plant breeders and seed companies Efforts: attended ASTA vegetable seed conference and had an exhibition booth describing our epigenetic technology. Spoke with industry participants about our technology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Participants improved their skills in plant breeding, field testing, molecular biology, plant transformation, and computational biology. How have the results been disseminated to communities of interest?We attended the ASTA vegetable and flower annual meeting, and set up a booth to described and discuss our technology with plant breeders and research directors. What do you plan to do during the next reporting period to accomplish the goals?We have made excellent progress on our objectives in year 1 and anticipate successfully fulfilling these objectives in year 2.
Impacts
What was accomplished under these goals?
Impact: we have discovered a new biological method that uses epigenetics to increase yields in tomatoes. Our field trial results demonstrate this can be used successfully in commercial production of tomatoes. Importantly, this method creates heterosis in tomatoes, which rarely display increased yields in hybrid progeny. Objective 1. 1A. 2017 Spring Florida Field Trial (the site was near Tampa in a town called Thonotosassa and the test was performed by Pacific Ag Research Corporation) Purpose: field test epigenetic effects on yield in first (S1), second (S2), and third (S3) generations after grafting of Rutgers (tomato variety we are using) shoots to an epigenetically improved Rutger's rootstocks. Data was from 3 replicate rows, where for each row only the inner 12 plants in a row of 21 to 27 plants were harvested. Conclusion: excellent yield improvements from the first, second, and third generation progeny (we count the number of generations after the graft to the epigenetic rootstock). This field trial demonstrates at least three generations of yield improvement post grafting are possible. 1B. 2018 Spring California Field Trial: S4 Graft Progeny (at Huasna in southeastern San Luis Obispo County, California, performed by Pacific Ag Research Corporation ) Purpose: field test fourth generation progeny (S4 after grafting of Rutgers shoots to epigenetically improved Rutgers variety rootstocks). Data was from 6 replicate rows, where for each row only the inner 10 plants in a row of 18 plants were harvested. Conclusion: epigenetic considerably increases fruit and yield during the first 5 weekly harvests of S4 plants. This higher early productivity can have considerable commercial value by getting tomatoes to market sooner in high value windows of opportunity before other tomatoes are available. 1C. Pennsylvania State University at State College: Fruit count for S4 lineages. (performed at the PSU field station in collaboration with Dr. Mackenzie) Purpose: field test fifth generation after the graft (S5) epigenetic lineages. Data was from 4 to 6 replicate rows, where for each row only the inner 14 plants in a row of 20 plants were harvested. Conclusion: The considerably higher fruit count per plant observed indicates the epigenetic effect from grafting persists for at least 5 generations. This is very helpful to know in order to describe the properties of our epigenetic system to our seed company partners. Objective 2. Purpose: determine epigenetic potential of individual mother plants grown from seeds from the grafted plant (S1 plants, which are the first generation of progeny from the graft). Each of lineages in the three population consists of 6 replicates each. Conclusion: 1) there is variation within individual S1 progeny mother plants in a population, although most individuals in the 26B population are above the average of the Rutgers control population; 2) it is possible to select and breed for individual lines within the population, to increase the overall fruit yields. Objective 3. Evaluate Phase I Msh1-RNAi Florida lines for competence as epigenetic rootstocks at their T2-T4 generations. Our transgenic Msh1-RNAi suppressed Florida tomato varieties initially had 'Msh1' phenotypes (variegated leaves and some abnormal phenotypes), which was reassuring that the lines were responding epigenetically as expected. But later generations of the lines (it takes a few generations to establish full epigenetic changes) predominantly became normal looking, which is a cause for concern as to whether they contain useful epigenetic changes. Concurrently, we obtained an EMS-derived genetic mutation in the tomato Msh1 gene from a TILLING collection. As this msh1(-) mutation encodes a stop codon in the reading frame of the Msh1 gene, it should be a null mutation. To summarize, due to the recent availability of msh1(-) null mutations that have improved technical properties and do not require regulatory approval (or consumer concerns about GMOs or gene-editing), we have switched our goal for objective 3 to focus on the EMS null mutation materials. These materials will be tested as epigenetically modified rootstocks in grafts that will be tested in field trials in 2019 to fulfill Objective 3. Objective 4. Computationally determine the methylome signatures of candidate epigenetic rootstocks (in collaboration, via a subaward, with the Mackenzie group now at Pennsylvania State University at University Park). Result 1. Our collaborator Dr. Sally Mackenzie has made excellent progress on improving computational programs for analyzing methylation changes in the genomes of epigenetically modified plants. Two 'pre-print' stage manuscripts are available on bioRxiv and the MethylIT computational program is available on R as described in reference 2 below: 1. Enhancing resolution of natural methylome reprogramming behavior in plants Robersy Sanchez, Xiaodong Yang, Hardik Kundariya, Jose Raul Barreras, Sally Mackenzie bioRxiv doi: https://doi.org/10.1101/252106 2. Example of Methylome Analysis with MethylIT using Cancer Datasets Robersy Sanchez, Sally Mackenzie doi: https://doi.org/10.1101/261982 Patents have been filed on the computational analysis method by the University of Nebraska, and Epicrop is the exclusive licensee for use in plants. We have installed the MethylIT computational program at Epicrop and are using it to analyze DNA methylation signatures in our epigenetically modified plants. The Mackenzie group has been able to distinguish epigenetically modified tomatoes and Arabidopsis plants using this MethylIT program. We have found it distinguished epigenetically modified soybean plants from control soybean plants and should be able to implement it in tomatoes as soon. These experiments benefit from proper genetic relationships of the biological materials as described below. Conclusion: excellent progress on a novel DNA methylation analysis computational program (named MethylIT) has been made by the Mackenzie group and this program is now available to us. Initial analyses by the Mackenzie group indicate this program can distinguish control from epigenetically improved plants. We will be implementing this in tomatoes in year 2. Objective 5. Determine yield performance of F1 epi-hybrids of genetically diverse parents, where one or two of the parents are epigenetically improved. 5A. 2018 Spring California Field Trial: Test of epigenetics in a F1 hybrid (at Huasna in southeastern San Luis Obispo County, California, performed by Pacific Ag Research Corporation ) Heterosis is defined as the progeny having higher yields than either parent. The tomato industry uses hybrids to obtain better genetics from two parents, rather than doing the extra breeding to fix all the genetics in a single inbred variety. They rarely observe heterotic yield increases in their F1 seeds as elite inbreds apparently have little heterotic combining ability. Confirming this. in our experiment, we also observed a lack of heterosis in our control cross of Rutgers x MoneyMaker, as the fruit count and yields are about the same as those of the parents. However, the epigenetic crosses had yield increases of 10 to 20% over the control crosses. Conclusion: We have created heterosis in tomatoes using our epigenetic technology. This is a stunning result of academic and commercial importance! Academically, this proves epigenetics alone can create heterosis, while still allowing for genetic contributions as well. In our case the lines are identical except for the epigenetic changes. Commercially, this increases yields in hybrid tomatoes, increasing value to both the seed companies and the growers. It is worth noting that this is our first attempt, and further improvements in the different genetic and epigenetic combinations are likely.
Publications
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