Progress 06/01/23 to 05/31/24
Outputs
Target Audience:We have shared our progress and approaches with industry and academic partners through the annual meeting of the Program of Excellence in Natural Rubber Alternatives at The Ohio State University. Changes/Problems:We requested and received a one-year no-cost extension, extending the project end date to May 2025. This additional time will allow us to fully address all the goals proposed in the project. What opportunities for training and professional development has the project provided?Our team member Camila Gutierrez Manriquez has gained valuable experience in planning and establishing evaluation nurseries, conducting phenotypic evaluations, managing samples for genotyping, and documenting seed characteristics. Camila has also gained experience in bioinformatics related to marker identification and preparation for analysis of mapping populations. How have the results been disseminated to communities of interest?Camila Gutierrez Manriquez has presented posters on germplasm evaluation and management at the Graduate Student Symposium of the Department of Horticulture and Crop Science at The Ohio State University and the Research Conference of the College of Food, Agriculture and Environmental Sciences at Ohio State. What do you plan to do during the next reporting period to accomplish the goals?Within Objective 1: By leveraging our genome data and population development, we plan to explore commercial platforms that may improve the cost-effectiveness of genotyping and screening for domestication genes. Within Objective 2: We have completed phenotypic evaluations of F2 and F3 populations, with genotyping data planned to be generated by the end of 2024. Deposit of materials in the repository in Pullman, WA has been delayed to provide fresher samples and is now planned for May 2025. We expect to receive data from the Community Science Program at the Department of Energy (DOE) Joint Genome Institute (JGI) by the end of 2024. This information should help improve indirect phenotyping and selection of germplasm. Within Objective 3: We will integrate our developed genetic markers with phenotypic information from evaluated germplasm to determine the most appropriate methods for adapting this information into a training population. This will ensure that our implementation of genomic selection effectively informs the genetic improvement of rubber dandelion.
Impacts
What was accomplished under these goals?
Throughout history, humans have domesticated crops to meet diverse needs including food, clothing, building materials, medicine, and chemical compounds. Thus, modern crops serve not only as food but also support industrial supply chains. For some materials essential to modern life, crop diversity is severely limited--sometimes restricted to a single species or geographic region--which creates significant supply risks. This is particularly true for natural rubber, which is currently produced by only one tree species (the rubber tree) grown overseas. Despite increasing domestic demand for natural rubber, production has decreased over the past decade due to numerous threats facing rubber trees and their habitats. To address this issue, our project aims to supplement the natural rubber supply chain (as well as other supply chains such as inulin) by developing a domestically grown crop: the rubber dandelion plant (Taraxacum kok-saghyz). Our impact will be based on providing useful varieties of this plant to farmers, industry partners, and researchers. These useful plant forms will include characterized and domesticated genetic material suitable for agriculture in temperate regions of the United States, with ongoing breeding to improve its utility in the short, medium, and long term. Our goal is to develop a rubber dandelion population that is cultivable, useful, and genetically diverse to ensure continued improvement. To achieve this, we're employing both well-established plant breeding techniques and cutting-edge genomic tools to accelerate the domestication of the rubber dandelion and establish a collection that meets the needs of American farmers and industry. Within Objective 1 Explore allelic diversity of domestication-related genes: Out of approximately 1,000 genetic polymorphisms previously identified using AmpSeq, we have processed and developed specific marker-trait associations to identify plants with desirable characteristics, including self-fertility, herbicide tolerance, tap root development, and non-biennial growth habit (flowering time of more than one season). However, cost-effectiveness and economies of scale remain challenging for genetic screening. We're combining these markers with the genome-wide markers identified during 2023 (see results for Objective 2) to create a comprehensive set of molecular markers. We're working with Agriplex Genomics to develop a commercial array that will make genotyping more efficient. Within Objective 2 Identify genetic components related to field performance and yield: In summer 2023, we established an evaluation nursery at The Ohio State University's CFAES Wooster campus. This nursery contains growing populations from eight distinct generations of our "alphabetical" breeding line and three generations from our "presidential" populations. We evaluated F2 populations and generated several F3 populations, though we observed inbreeding depression in some crosses. Genotyping of these populations is planned for late 2024. We selected 54 half-sibling families from our latest generations (Juliette and Jefferson) based on field performance. After harvest and drying, these samples were analyzed using near-infrared spectroscopy (NIR). We extracted DNA from each half-sibling family and identified genome-wide single nucleotide polymorphisms (SNPs) using our reference genome OH-1004 (NCBI accession: JAJTUU000000000). This analysis involved sequencing data from 30 individuals (prepared in three independent genomic libraries) at 15X coverage for each generation. From more than 2 million SNPs identified, we've selected approximately 5,000 as most informative and relevant for high-throughput genome-wide genotyping. Within Objective 3 Define core germplasm to implement genomic selection for the improvement of complex traits: Through the genotyping of different generations from our alphabetical and presidential populations, the publication of the OH-1004 genome, and subsequent gene prediction and annotation, we have identified high-quality SNPs. These genetic markers, together with the phenotypic information collected from these populations, will serve as a training dataset to define our core germplasm collection.
Publications
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The progress made so far in the project and the approaches being adapted and implemented have been shared with industry and academic partners through the annual meeting of the Program of Excellence in Natural Rubber Alternatives at The Ohio State University and the National Association of Plant Breeders' 2022 Annual Meeting, in Ames, Iowa. These meetings were attended by industry stakeholders, researchers, and graduate students. Changes/Problems:Understaffing has been the main limiting factor for making progress in the project in aspects regarding the field. Inclement weather during June and July 2022 negatively impacted the increase of germplasm and the operation of some facilities. Phenotyping through Accelerated Solvent Extraction (ASE) has shown to be device-dependent, very variable, and non-cost-effective, so efforts in phenotyping using this technique will be replaced by the use of near-infrared spectroscopy (NIR). The replacement of the graduate students supported by the project has successfully occurred, but some delay has meant that a non-cost extension for the project was requested so the goals proposed in the project can be addressed in full. What opportunities for training and professional development has the project provided?This project has enabled master's student Camila Gutierrez Manriquez to learn about the production of mapping populations and the analysis of traits for the implementation of marker-assisted selection. How have the results been disseminated to communities of interest?Camila Gutierrez Manriquez, the MS student supported through this project, has presented posters on the development of mapping populations in the Graduate Student Symposium of the Department of Horticulture and Crop Science at The Ohio State University and the Research Conference of the College of Food, Agriculture and Environmental Sciences at Ohio State. The latter is a conference open to the general public. Also, Camila had the opportunity to present a poster and earned an award at the National Association of Plant Breeders' 2022 Annual Meeting in Ames, Iowa, in which plant breeders and students were the main audiences. What do you plan to do during the next reporting period to accomplish the goals?A one-year no-cost extension was requested, so the current end date of the project is May 2024. Objectives 1 and 2 are being emphasized, and tools are being deployed to secure progress on aspects related to Objective 3. Within Objective 1: Scaling the AmpSeq pipeline aided by the newly gene-annotated genome assembly will enable high-throughput genotyping and production of molecular markers for marker-assisted selection (MAS). The use of a distinct platform (e.g., PacBio) may end up outside of the current availability of funding, but the scrutiny of additional genes on targeted relevant germplasm will be pursued. Within Objective 2: Screening of several F2 populations will enable a phenotypic evaluation with an emphasis on self-compatibility and herbicide tolerance. F3 populations will be also developed. Selection in the field and increase of seed of distinct generations of germplasm will be pursued. It is expected that by early 2024 genotypic data will be actively analyzed to identify marker-trait associations. Conversations have been started with the germplasm repository in Pullman, WA, to set the deposit of accessions of germplasm that may be of interest to the community. An increase of seed from such germplasm accessions will be performed during 2023 and the beginning of 2024 with the purpose of sharing seed with the repository by May 2024. Given the issues to genotype rubber concentration using Accelerated Solvent Extraction (ASE), support from the Community Science Program at the Department of Energy (DOE) Joint Genome Institute (JGI) has been pursued through a New Investigator support grant in order to evaluate whether targeted metabolomics is an option to phenotype germplasm for biopolymer content. The experiment will take place using germplasm included in the current project during Autumn 2023. Within Objective 3: The first complete gene prediction and functional annotation of predicted genes are expected to be concluded by early Autumn 2023, and the release of this annotated version of the TK genome will be done through NCBI as an update of the accession number: JAJTUU000000000. Transcriptomic data will be also released through the Sequence Read Archive (SRA). These resources will be beneficial to make progress on Objectives 1 and 2, which will set the foundation for Objective 3. The development of a pseudomolecule-level assembly and functionally annotated genes genome sequence will provide a strong foundation for the identification of relevant polymorphisms and gene sequence variants for analysis of the breeding germplasm developed so far.
Impacts
What was accomplished under these goals?
Throughout history, humans have domesticated crops to meet such diverse needs as food, clothing, building materials, medicine, and chemical compounds. Thus, modern crops are not restricted to simply food but can meet the needs of industrial supply chains. For some of the materials necessary for modern life, the diversity of crop sources is very limited, sometimes restricted to a single species or geographic region, which introduces supply-side risk. This is true for natural rubber, a material that is produced by only one tree species, the rubber tree, which is grown overseas. Despite the increasing domestic demand for natural rubber, production has decreased over the past ten years due to the myriad threats facing the rubber tree and its habitat. To address this issue, this project aims to supplement the natural rubber supply chain (as well as other supply chains such as inulin) through the development of a domestically grown crop. This impact will be based on providing useful forms of the rubber dandelion plant (Taraxacum kok-saghyz) to farmers, industry, and researchers. Useful plant forms will include characterized and domesticated genetic material for agriculture in the temperate regions of the US and continued breeding to improve its utility in the short, medium, and long terms. Our goal is a rubber dandelion population that is cultivable, useful, and diverse to ensure its further genetic improvement. To this end, this project employs both well-established techniques in plant breeding and new tools derived from genomics to accelerate the domestication of the rubber dandelion and thus establish a collection of rubber dandelions to meet the needs of American farmers and industry. Within Objective 1 Explore allelic diversity of domestication-related genes: Using the pipeline AmpSeq, originally developed for grapevine, genotyping of 96 genes from 96 samples has been performed. The heterogeneous amplification among genes and samples was a major issue, so this procedure has shown more promise for scrutinizing a large number of genes and samples in practice. Approximately 1000 polymorphisms have been identified in approximately 70 gene sequences. These variants are being filtered for quality and reliability for subsequent analysis. Planning for additional genotyping of the rest of the domestication-related genes is ongoing. Long genes (beyond 600 bp) have shown to be problematic to amplify as well as to assemble. The design of more specific primers has been one of the first approaches; however, a change to an amplicon sequence platform using long-read sequencing (e.g., PacBio) is another option. For this endeavor, scalability is the main concern to make this approach cost-effective for sequence variant discovery. Subsequently, the design and implementation of genotyping platforms, such as agriplex, will be a more efficient option for genotyping for selection and breeding purposes. Within Objective 2 Identify genetic components related to field performance and yield: In summer 2022, the establishment of an evaluation nursery of growing populations from eight distinct generations of selection was performed in the fields of the CFAES Wooster campus of The Ohio State University. Although the number of plants recovered for the first four generations was minimal, and there were issues of germination and establishment of populations from generations 6 and 7, an increase of plants and seeds for each generation was possible, as well as selection of individuals with more than 10 g fresh weight root mass in the 8th generation. DNA samples from the recovered plants in the field have been saved, and genotyping is being planned. Unfortunately, phenotyping information has been difficult to produce on these plants since accelerated solvent extraction (ASE) of natural rubber has not been reliable, and determination through near-infrared spectroscopy (NIR) has not been possible since the equipment has been under maintenance. However, data on root mass is available, and a replication of the nursery will be attempted during summer 2023. The regeneration of germplasm for phenotyping and mapping that was lost during lockdown has been of the highest priority, and the resynthesis of F1 populations was successful, although behind schedule, and the recovery of F2 seed is ongoing, expecting screening and growth of plants in autumn 2023. Within Objective 3 Define core germplasm to implement genomic selection for the improvement of complex traits: Gene prediction and annotation of the inbred line of TK started by producing transcriptomic data from tissues including roots, rosettes, flowers, and seeds. This data has been used to perform de novo transcriptome assembly, and it is being used to feed and inform gene prediction
Publications
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The progress made so far in the project and the approaches being adapted and implmentedhave been shared with industry and academic partners through the annual meeting of the Program of Excellence in Natural Rubber Alternatives at The Ohio State University, and the TechnoConnect World Innovation Conference and Expo in Washington D.C.These meetings were attended by industry stakeholders, researchers and graduate students. Changes/Problems:COVID-19 has continued impacting the health of personnel. Thus, the Ph.D. student who began to work on this project halted graduate school, which left the project without a main driver for more than six months in 2021. Understaffing has been the main limiting factor for making progress in the project, given the long times required to hire personnel or recruit students.This has severely affected germplasm maintenance and the generation of phenotypic data. In addition, a facilities malfunction that led to an issue in greenhouse temperature regulation caused the loss of more than 1000 plants ofT. kok-saghyz,representing several mapping populations of germplasm freshly regenerated and relevant to this project.Major changes in the plan include the replacement of the graduate students supported by the project, as well as a redirection of efforts and funds to produce a high-quality genome assembly to guide efforts to meet the goals proposed in the project. What opportunities for training and professional development has the project provided?This project has enabled the training of a visiting scholar regarding the scrutiny of gSSRs inTaraxacum kok-saghyzgermplasm germplasm. Subsequently, a summer intern had the opportunity to work on the regeneration of germplasm and inventory of seeds of self-seeding lines. Since January 2022, a master's student has integrated into the project, recovering the aspects regarding production of mapping populations with germplasm with self-seeding and herbicide tolerance traits. How have the results been disseminated to communities of interest?Progress made and perspectives have been shared through the annual meeting (March 15, 2022) of the Program of Excellence in Natural Rubber Alternatives at OSU. This meeting is attended by industry partners (Goodyear Tire and Rubber Co. and American Sustainable Rubber LLC.) and academic partners from the University of Akron, the University of Nebraska, and Oregon State University. Additionally throughthe TechnoConnect World Innovation Conference and Expo in Washington D.C. on October 19, 2021, there was an opportunity to reach stakeholders involved in domestic natural rubber esearch and development. Camila Gutierrez Manriquez, the MS student supported through this project, has presented posters on the development of mapping populations in the Graduate Student Symposium of the Department of Horticulture and Crop Science at The Ohio State University and the Research Conference of the College of Food, Agriculture and Environmental Sciences at Ohio State. The latter being a conference open to the general public. Jonathan Fresnedo Ramirez, PD of the project, was invited to give a virtual presentation on the domestication ofTaraxacum kok-saghyzin the Corteva- Plant Breeding, Genetics, and Biotechnology (PBGB) Symposium - 'Orphan Crop Breeding for Diverse and Sustainable Food Options' (organized by graduate students) at East Lansing, MI. December 10, 2021.https://www.canr.msu.edu/pbgb/Symposium/. Similarly, he was also invited to give a virtual seminar on the Genomics-aided research in the domestication and breeding of outcrossing crops, in which work onT. kok-saghyzwas emphasized. What do you plan to do during the next reporting period to accomplish the goals?We estimate that we are currently about a year behind the original schedule provided. We have updated this timeline and likely will request one year of non-cost extension. Objectives 1 and 2 will be emphasized, and we are confident that we will make progress on aspects related to Objective 3. WithinObjective1: Tailoring the AmpSeq pipeline using the new genome assembly will enable high-throughput genotyping and production of molecular markers for marker-assisted selection (MAS). The process basically involves the use of robotics to enable a high-multiplexing of PCR reactions, tweaking the existing scripts for amplicon analysis, identifying genetic polymorphisms in the germplasm, marker-trait association, discovery of variants associated with desired phenotypes, and the design of molecular markers for MAS. WithinObjective2: The production of several F2populations will enable a phenotypic evaluation with emphasis on selfing-herbicide tolerance; however other traits will be scrutinized. Investment in being able to quantify rubber through accelerated solvent extraction will be done on distinct germplasm sections, including otherTaraxacumspecies. The graduate student supported through the project will focus on the performance of analysis of genotypic and phenotypic data to discover marker-trait associations that enhance the selection ofT. kok-saghyz.
Impacts
What was accomplished under these goals?
Throughout history, humans have domesticated crops to meet such diverse needs as food, clothing, building materials, medicine, and chemical compounds. Thus, modern crops are not restricted to simply food but can meet the needs of industrial supply chains. For some of the materials necessary for modern life, the diversity of crop sources is very limited, sometimes restricted to a single species or geographic region, which introduces supply-side risk. This is true for natural rubber, a material that is produced by only one tree species, the rubber tree, which is grown overseas. Despite the increasing domestic demand for natural rubber, production has decreased over the past ten years due to the myriad threats facing the rubber tree and its habitat. To address this issue, this project aims to supplement the natural rubber supply chain (as well as other supply chains such as inulin) through the development of a domestically grown crop. This impact will be based on providing useful forms of the rubber dandelion plant (Taraxacum kok-saghyz) to farmers, industry, and researchers. Useful plant forms will include characterized and domesticated genetic material for agriculture in the temperate regions of the US and continued breeding to improve its utility in the short, medium, and long terms. Our goal is a rubber dandelion population that is cultivable, useful, and diverse to ensure its further genetic improvement. To this end, this project employs both well-established techniques in plant breeding and new tools derived from genomics to accelerate the domestication of the rubber dandelion and thus establish a collection of rubber dandelions to meet the needs of American farmers, industry, and healthcare. WithinObjective1 Explore allelic diversity of domestication-related genes, 2) identifygenetic components related to field performance and yield: The approach based on amplicon sequencing using the Fluidigm platform for PCR multiplexing proved to be impractical, and its implementation was not cost-effective, even after several rounds of customization that included bioinformatics and wet lab modifications. Development of scripts that enableamoreefficientandtargeted design of primers for amplicons of genes of interest has been pursued, and it is currently being applied to two hundred genes related to aspects of domestication and rubber production. These scripts are available and are maintained in the GitHub repositoryhttps://github.com/Fresnedo-Lab/MPAS. WithinObjective2 Identifygenetic components related to field performance and yield: In the continuation of the testing of gSSRsreported by Nowicki et al.(citation: Nowicki et al., 2019, Scientific Reports) within the OSU germplasm, itwas found thatonly15gSSRsintermittently amplified, Tar4, Tara7, Tara8, Tara11, Tara22, Tara23, Tara26, Tara27, Tara29, Tara32, Tara41, Tara43, Tara39, and Tara46. The expected fragment size for each of these gSSRs corresponded to previous reports in literature. The recovery of germplasm lost during the COVID-19 lockdown has been slow, and it has also been affected by issues with facilities. Thus, the accessions regenerated for self-seedinglines, parental lines for forming segregating populations, divergent germplasm for germination at low temperature, and populations of relative species got lost in a greenhouse malfunction. Emphasis has been placed on increasing seed in self-seeding lines and resynthesizing heterozygous lines for mapping population production.The regeneration of germplasm for phenotyping and mapping has been of the highest priority. Resynthesis of F2 populations has been possible, and the germplasm is being grown in two distinct greenhouses. Currently, 2600 seeds of resynthesized F1seed have been produced, and approximately 1000 seeds of F2mapping populations are being processed for planting and screening in Autumn 2022. Given the issues with the genotyping methods that are pivotal for Objective 1 and Objective 3, we shifted efforts towards producing a high-quality genome sequence from the self-seed accession produced after eight generations, which has been successfully completed. The assembly of this genome data provides an assembly at the pseudomolecule level representing 1.25 Gb of the TK genome and is a pivotal resource to customize and guide subsequent efforts for the development of high-throughput genotyping. This genome will be released publicly through NCBI with the accession number: JAJTUU000000000.
Publications
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:Scientists,farmers,processers,manufacturers,therubberindustry. Changes/Problems:COVID-19 impacted the health of multiple personnel. One technician resigned which, coupled with a university-imposed hiring pause, which left the project understaffed until staff could be hired to cover positions. This is severely affecting the generation of phenotypic data, for example. The associated lockdowns resulted in restricted access to core facilities, loss of germplasm, and no access to wet lab facilities until three months after the official start of the program. Based on current epidemiological trajectories and management of access to facilities we find ourselves getting back on track with work and expect that we will be able to catch up to a large extent during 2021. No major changes are currently expected in the work plan. What opportunities for training and professional development has the project provided?Daniel Williams, the PhD student involved in the development of this project, has an interdisciplinary background encompassing plant science, archaeology, and computer science. This project has enabled Daniel to get involved into operations and logistics of a plant breeding/domestication program. Thus, Daniel has actively being involved in aspects of information management, such as database design, genotyping design, computing, phenotyping, and plant management. Additionally, Daniel Williams presented at two symposia: The Society for Economic Botany Fall 2020 Symposium (October 23, 2020) and the 14th Annual Horticulture and Crop Science Graduate Research Symposium (December 15-16, 2020) at the Ohio State University. The Graduate Research Symposium was held online for the first time this year and was attended by students, faculty, and staff of the Ohio State university Department of Horticulture and Crop Science. Daniel presented an overview of neodomestication concepts, an updated conceptual model for neodomestication, and updates to the AmpSeq pipeline for identification of genetic polymorphisms. How have the results been disseminated to communities of interest?The progress made so far in the project has been shared through the annual meeting (March 9, 2021) of the Program of Excellence in Natural Rubber Alternatives at OSU. This meeting is attended by industry partners (Goodyear Tire and Rubber Co. and American Sustainable Rubber LLC.) and academic partners from University of Akron, University of Nebraska and Oregon State University. Daniel Williams gave a presentation in the Society for Economic Botany Fall 2020 Symposium (October 23, 2020). This symposium was online and brought together practitioners, researchers, and students from at least five continents. Daniel Williams provided an overview of concepts of neodomestication and its implementation inTaraxacum kok-saghyzusing genomics approaches in a technoeconomics perspective. A recording of the symposium can be found here: https://youtu.be/G4rOqlaGKCM. The agenda is posted here: http://www.econbot.org/file.php?file=sitefiles/Meetings/SEBFall2020SymposiumAgenda.pdf What do you plan to do during the next reporting period to accomplish the goals?We estimate that we are currently about six months behind the original schedule provided. We have updated this timeline and are confident that we will be able to advance quickly in many of the task within the first two objectives of the project. Below, plans are provided. Within Objective 1: Implementation of the improved AmpSeq pipeline and generation of results will enable production of molecular markers for marker-assisted selection (MAS). The process will involve tweaking the existing scripts for amplicon design, identifying genetic polymorphisms in the germplasm, marker trait association, discovery of variants associated with desired phenotypes, and the design of molecular markers for MAS. Validation and optimization of the markers will begin next year and will enable tasks delineated in Objective 3 (definition of core germplasm sets). Within Objective 2: The regeneration of germplasm for phenotyping and mapping of traits is the highest priority. The goal is to resynthesize (at least) the F2 populations for combination of selfing-herbicide tolerance. Additional crosses are being performed, including interspecific hybridizations with other Taraxacum species, specifically T. linearisquameum, to ease the interrogation of traits related to plant vigor. It is expected that an additional graduate student will join to this project with a research focused on differentiating obligated outcrossers from selfing plants. Given the complications to obtain DNA of enough quality for genotyping-by-sequencing (GBS) approaches. We are transition to implement a technique based on PCR amplification (GBS is based on DNA digestion) that will enable massive genotyping of germplasm, this technique is K-seq (citation: Ziarsolo et al, 2021, Plant Methods). It is one of the main emphases for the next year of work, and what will enable us to interrogate mapping populations and approach the tasks delineated in objective 3 (definition of core germplasm sets).
Impacts
What was accomplished under these goals?
Crops are one of the many ways in which humanity has innovated to manage scarcity of key resources, such as food, clothing, building materials, medicine, and chemical compounds. Modern crops are sources of valuable materials beyond nutrition. For some of these materials the diversity of crop sources is very limited, sometimes restricted to a single species or geographic region, which imposes supply-side risks to meeting societal and industrial needs. This is true for natural rubber, a material which is critical for industry and health care. Natural rubber is produced by only one tree species, the rubber tree, which is grown overseas, and which faces diverse constraints and threats that have forced production to decrease over the last ten years despite increasing domestic demand. In this vein, this project aims to make an impact by enabling the development of a domestically grown crop that will supplement the natural rubber supply chain (and by extension the inulin supply chain because both chemical compounds are produced by the plant that is the subject of this project). This impact will be based on providing useful forms of the rubber dandelion plant (Taraxacum kok-saghyz) to farmers, industry, and researchers. Useful plant forms will include characterized and domesticated genetic material for both agriculture in the temperate regions of the US and continued breeding to improve its utility in the short, medium, and long terms. To do that, this project employs tools from genomics and well-established techniques in plant breeding to accelerate the domestication of the rubber dandelion and thus establish a collection of rubber dandelions that is cultivable, useful, and diverse to ensure its further genetic improvement to meet the needs of American farmers, industry, and healthcare. Within Objective 1 Explore allelic diversity of domestication-related genes: Our original approach of using the Fluidigm®platform for PCR multiplexing proved to be inflexible when we tried to implement amplicon sequencing on numerous samples with very heterogenous DNA quality. Therefore, we have developed some customizations to our AmpSeq pipeline (citation: Fresnedo Ramirez et al, 2019, Frontiers in Plant Science). Switching from Fluidigm to a robotically controlled PCR process, which has relaxed amplicon and primer design parameters and DNA quality requirements, improves expected DNA extraction, amplification, and sequencing efficiency. Doctoral student Daniel Williams has implemented the following customizations to the Primal Scheme pipeline (citation: Quick et al, 2017, Nature Protocols): a script written for the R statistical language formats gene sequence data into fasta files for Primal Scheme; a shell script to run Primal Scheme on all genes with specified primer design parameters (optimized for use in the Ohio Supercomputer Center); two R scripts R to retrieve data from Primal Scheme and to produce a visual report; a shell script to consolidate newly designed primers into an easily readable format; an R script to sort primers by pool and format as fasta files; a shell script to run a local installation of Clustal_Omega (citation: Madeira et al, 2019, Nucleic Acids Research) separately on all pools; and an R script to melt the identity matrix into a table of easily readable pairwise comparisons to aid pool designs and optimization. Additionally, a script for automated design of optimal PCR pools has been specified. These custom scripts enable a customized pipeline for a more efficient and targeted design of primers for amplicons of genes of interest. These scripts are available in the GitHub repository https://github.com/Fresnedo-Lab/MPAS. We expect some other genes related to domestication and for other relevant traits can be applied to the pipeline as we proceed. The custom process produced 1098 pairs of primers for 1098 predicted amplicons representing eighty-one percent of the sequence length of the 103 targeted genes. The primers ranged in length from 21 to 33 bp (median = 22), in percent GC content from 30 to 55 (median = 50), and in melting temperature from 59.5 to 62.2 ºC (median = 61.0 ºC). Within Objective 2 Identify genetic components related to field performance and yield: The gSSRs reported by Nowicki et al (citation: Nowicki et al, 2019, Scientific Reports) have been tested in several accessions within a pedigree. Eleven gSSRs appear to be polymorphic in the tested accessions. These are at loci Tara04, Tara08, Tara11, Tara14, Tara22, Tara23, Tara26, Tara27, Tara29, Tara32, Tara41, and Tara46, according to Nowicki et al. During the COVID-19 lockdown germplasm was lost. We are in a stage of germplasm regeneration focusing on self-seeding lines, parental lines for forming segregating populations, and maintenance of diverse accessions which may contain valuable genetic diversity. Currently growing are 397 self-seeding plants of three independent lines; 31 seventh generation protoporphyrinogen oxidase (PPO) inhibiting herbicide tolerant plants, 35 plants from the seventh generation of a population selected for vigorous growth, 526 plants representing the diversity of five distinct Taraxacum species, and hundreds of additional plants associated with ongoing experiments in germination and survival related to seasonal conditions.One of the self-seeding lines is previously unreported. This line, internally named "OH-205" was selected from a population recursively selected for vigor and appears to exhibit faster germination and more robust growth than the previously identified selfing lines. Given limited access to facilities, phenotyping was conducted the effect of ground temperature on six different categories of germplasm during germination. The measured traits included: germination rate, days to emergence, days to first true leaf, and days to second true leaf. Results show that more advanced populations exhibit more vigorous seedlings, earlier germination, germination at lower temp, and increased uniformity of germination time. There is a ground temperature limit at 6 ºC at which germination is very low. Seedlings that germinated at low temperature and those that germinated after exposure to higher temperatures have been separated for future characterization, selection, and introduction into the germplasm improvement pipeline.
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