Progress 05/15/19 to 05/14/22
Outputs
Target Audience:The target audience for this project included Cornell University graduate students, the maize genetics community, and U.S. sweet corn stakeholders including researchers, private and public breeders, and seed companies. Efforts: PD Hershberger was trained in the analysis of transcriptomic data for association and prediction analyses. PD Hershberger presented the results of this project in poster form at the National Association of Plant Breeders annual meeting in August 2021 and through an in-person oral presentation to stakeholders at the International Sweet Corn Development Association meeting in December 2021. A peer-reviewed manuscript describing the results of this project in depth was published in The Plant Genome Journal in 2022. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities provided to PD Hershberger by the project include one-on-one training related to the quality control and analysis of transcriptomic datasets, the development of reproducible analysis pipelines, and statistical genetics techniques such as genome- and transcriptome-wide association and genomic prediction. Additional training in writing, revising, editing, peer review, presentation development, and public speaking were provided through the dissemination of project results. Although many in-person professional development opportunities were not available during this reporting period due to COVID-19, PD Hershberger presented work related to this project in poster form at the virtual National Association of Plant Breeders annual meeting in August 2021. PD Hershberger also presented the results of this project via an in-person talk at the International Sweet Corn Development Association Annual Meeting in Chicago, IL in December 2021 and virtually through her defense seminar in June 2021. Beyond conferences, Cornell University offers a wide variety of relevant seminars, many of which PD Hershberger attended during this reporting period. PD Hershberger also attended individual and group meetings with seminar speakers to network andlearn more about their work. Ultimately, this project played a major role in the development of PD Hershberger as a scientist. The skills gained and relationships built through this project played a major role in PD Hershberger's job search after graduation with her Ph.D. She has now started as an Assistant Professor of Vegetable Breeding and Genetics at Clemson University and hopes to continue to contribute to improving the nutritional quality of vegetables for production and consumption in the United States. How have the results been disseminated to communities of interest?Results from this project have been disseminated widely both within and outside of the sweet corn community. A peer-reviewed journal article was published in The Plant Genome Journal in 2022. Project results were shared through PD Hershberger's defense seminar at Cornell University in June 2021 and at the 2021 International Sweet Corn Development Association Annual Meeting in Chicago, IL. A poster detailing the results of this project was presented virtually at the National Association of Plant Breeders 2021 annual meeting. The following abstract was used to describe the work conducted through this project in the Plant Genome Journal and for both oral presentations: "Sweet corn (Zea mays L.) is consistently one of the most highly consumed vegeta- bles in the United States, providing a valuable opportunity to increase nutrient intake through biofortification. Significant variation for carotenoid (provitamin A, lutein, zeaxanthin) and tocochromanol (vitamin E, antioxidants) levels is present in temperate sweet corn germplasm, yet previous genome-wide association studies (GWAS) of these traits have been limited by low statistical power and mapping resolution. Here, we employed a high-quality transcriptomic dataset collected from fresh sweet corn kernels to conduct transcriptome-wide association studies (TWAS) and transcriptome prediction studies for 39 carotenoid and tocochromanol traits. In agreement with previous GWAS findings, TWAS detected significant associations for four causal genes, β-carotene hydroxylase (crtRB1), lycopene epsilon cyclase (lcyE), γ-tocopherol methyltransferase (vte4), and homogentisate geranylgeranyltransferase (hggt1) on a transcriptome-wide level. Pathway-level analysis revealed additional associations for deoxy-xylulose synthase2 (dxs2), diphosphocytidyl methyl erythritol synthase2 (dmes2), cytidine methyl kinase1 (cmk1), and geranylgeranyl hydrogenase1 (ggh1), of which, dmes2, cmk1, and ggh1 have not previously been identified through maize association studies. Evaluation of prediction models incorporating genome-wide markers and transcriptome-wide abundances revealed a trait- dependent benefit to the inclusion of both genomic and transcriptomic data oversolely genomic data, but both transcriptome- and genome-wide datasets outperformed a priori candidate gene-targeted prediction models for most traits. Altogether, this study represents an important step toward understanding the role of regulatory variation in the accumulation of vitamins in fresh sweet corn kernels." What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Despite the importance of vitamin E and provitamin A for human health and well-being, dietary intake of these vitamins is suboptimal in the United States. Sweet corn, which consistently ranks among the most highly consumed vegetables in the United States, is amenable to breeding for increased nutritional quality, providing an opportunity to meet these dietary needs through biofortification. Through this project, we generated and analyzed an RNA transcript abundance dataset from a diverse panel of sweet corn inbred lines to better understand the genetic control of these important vitamin traits. Using statistical genetics approaches, we identified seven causal genes involved in the biosynthesis and retention of the compounds responsible for vitamin E and provitamin A in fresh sweet corn kernels, two of which had not previously been identified in sweet corn. We also evaluated 13 model types for accuracy in the prediction of these vitamin traits. Despite moderate to high prediction accuracy across all models, the inclusion of transcript abundance data alongside existing genomic data only improved prediction accuracy for some traits. This project has provided a better understanding of the genetics underlying nutritional traits in fresh sweet corn kernels and developed tools for plant breeders to apply this knowledge to the generation of more nutritious sweet corn varieties. The results of this project have been disseminated widely through a peer-reviewed manuscript, conference presentations, and a Ph.D. dissertation. Data and code related to project activities have been made publicly available to maximize reproducibility. All objectives were completed during the previous reporting period, but results were disseminated during the current reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Hershberger, J., Tanaka, R., Wood, J. C., Kaczmar, N., Wu, D., Hamilton, J. P., DellaPenna, D., Buell, C. R., & Gore, M. A. (2022). Transcriptome-wide association and prediction for carotenoids and tocochromanols in fresh sweet corn kernels. Plant Genome, e20197. https://doi.org/10.1002/tpg2.20197
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Hershberger JM, Tanaka R, Wood JC, Kaczmar N, Wu D, Hamilton JP, DellaPenna D, Buell CR, Gore MA. Transcriptome-wide association and prediction for carotenoids and tocochromanols in fresh sweet corn kernels. Talk presented at the International Sweet Corn Development Association Annual Meeting. 2021 December 6, Chicago, IL
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Hershberger JM, Tanaka R, Wood JC, Kaczmar N, Wu D, Hamilton JP, DellaPenna D, Buell CR, Gore MA. Transcriptome-wide association and prediction for carotenoids and tocochromanols in fresh sweet corn kernels. Poster presented at the National Association of Plant Breeders Annual Meeting. 2021 August 16, Virtual
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Hershberger, J. 2021. Quality trait prediction in cassava and sweet corn. (Publication No. 28649779) [Doctoral dissertation, Cornell University]. ProQuest Dissertations and Theses
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Progress 05/15/20 to 05/14/21
Outputs
Target Audience:The target audience for this audience included Cornell University graduate students and the maize genetics community. Efforts:Both PD Hershberger and another plant breeding and genetics graduate student were trained in the analysis of transcriptomic data for association and prediction analyses. PD Hershberger presented a posterat the annual Maize Genetics Meeting in June 2020 describing the goals of the project and progress to date. Changes/Problems:Due to COVID-19, laboratory-based RNA extractions were delayed several months. Despite this delay, PD Hershberger was able to complete the extractions in July 2020 and submit the samples for sequencing as planned. Additionally, several samples were contaminated by the sequencing facility, reducing the number of samples available for analysis. After removingthe unusable samples, a sufficient number of samples were still of high quality and were used for analysis without further problems. What opportunities for training and professional development has the project provided?Training opportunities provided by the project include one-on-one training related to the quality control and analysis of transcriptomic datasets, the development of reproducible analysis pipelines, and statistical genetics techniques such as genome- and transcriptome-wide association and genomic prediction. Both PD Hershberger and a first-year graduate student were trained. Although in-person professional development opportunities were not available during this reporting period due to COVID-19, PD Hershberger presented work related to this project at the virtual Maize Genetics Conference in June 2020. PD Hershberger also participated virtually in the National Association of Plant Breeders annual meeting in August 2020. Beyond conferences, Cornell University offers a wide variety of relevant seminars, many of which PD Hershberger attended during this reporting period. PD Hershberger also attended individual and group meetings with seminar speakers to network and learn more about their work. How have the results been disseminated to communities of interest?Work related to this project was presented at the Maize Genetics Conference in June 2020, but results were not available until the very end of this reporting period so could not yet be shared with the broader community. These results will be further communicated in the next reporting period through conferences and manuscripts as described below. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, a manuscript describing the methods and findings resulting from the project objectives will be submitted to a peer-reviewed journal. This manuscript has been drafted and is undergoing internal revision by co-authors. This work will also be presented in poster form at the National Association of Plant Breeders annual meeting (virtual; abstract accepted) in August 2021. Additionally, PD Hershberger has been invited to speak about this project at the Land Institute in Salina, KS, later this summer.
Impacts
What was accomplished under these goals?
Despite the importance of vitamin E and provitamin A for human health and well-being, dietary intake of these vitamins is suboptimal in the United States. Sweet corn, whichconsistently ranks among the most highly consumed vegetables in the United States, is amenable to breeding for increased nutritional quality, providing an opportunity to meet these dietary needs through biofortification. Through this project, we generated and analyzed an RNA transcript abundance dataset from a diverse panel of sweet corn inbred lines to better understand the genetic control of these important vitamin traits. Using statistical genetics approaches, we identified seven causal genes involved in the biosynthesis and retention of the compounds responsible for vitamin E and provitamin A in fresh sweet corn kernels, two of which had not previously been identified in sweet corn. We also evaluated 13 model types for accuracy in the prediction of these vitamin traits. Despite moderate to high prediction accuracy across all models, the inclusion of transcript abundance data alongside existing genomic data only improved prediction accuracy for some traits. This project has provided a better understanding of the genetics underlying nutritional traits in fresh sweet corn kernels and developed tools for plant breeders to apply this knowledge to the generation of more nutritious sweet corn varieties. Objective 1: Collect novel gene expression data on a diverse panel of fresh sweet corn kernels This objective was completed during this reporting period. In June and July 2020, PD Hershberger isolated RNA from fresh sweet corn kernel samples that had been collected from a sweet corn inbred line diversity panel the previous summer (2019). Quality control was performed, and five plates (460 high quality samples, including checks) were submitted to the Cornell sequencing facility for library preparation and 3′ mRNA-seq. Resulting sequence reads were aligned to the B73v4, PH207, and Ia453 (sweet corn) reference genomes. Aligned reads were normalized and thorough quality control was performed, ultimately resulting in a high-quality dataset of 355 experimental samples. Best linear unbiased estimators (BLUEs) were calculated using the normalized transcript abundances for each gene passing quality control to account for field variation and sequencing plate effects, and probabilistic estimation of expression residuals was used to remove hidden factors (e.g., experimental noise) in further preparation for downstream analyses. The number of genes in the final BLUE datasets varied across genomes, with 18,765 genes in the B73v4 dataset, 17,922 for PH207, and 18,477 genes for the Ia453 alignment. Key outcomes from the completion of this objective include improved skills of PD Hershberger related to RNA extraction and the processing of high-dimension datasets. The data generated from the completion of this objective will be featured in at least one publication and will be released upon publication. Objective 2: Integrate these data with existing single-nucleotide polymorphism marker and fresh sweet corn kernel metabolite datasets through a combination of genome-wide association studies and transcriptome-wide association studies Transcriptome-wide association studies (TWAS) were performed using the high-quality normalized transcript abundance dataset generated in Objective 1 paired with existing fresh kernel tocochromanol and carotenoid phenotypic datasets from previously published studies of the same panel of diverse sweet corn inbred lines. A total of 44 models were compared for each of the 39 individual tocochromanol and carotenoid traits to optimize the number of principal components and evaluate whether a kinship term should be included in the model. As previous studies have found kernel endosperm mutation type (su1, sh2, or both) to be significantly associated with tocotrienol and some carotenoid traits, the inclusion of kernel mutant type as a fixed effect was also evaluated. Raw P-values were adjusted to control the false discovery rate (FDR), and those passing a 5% FDR threshold were considered significant. At a transcriptome-wide level, 23 unique genes passed this threshold, including three a priori candidate genes: β-carotene hydroxylase 1 (crtRB1), lycopene ε-cyclase (lcyE), and vitamin E synthesis4 (vte4). At a pathway level, additional significant associations were identified with four additional a priori candidate genes: homogentisate geranylgeranyl transferase1 (hggt1), diphosphocytidyl methyl erythritol synthase2 (dmes2), deoxy-xylulose synthase2 (dxs2), and cytidine methyl kinase1 (cmk1). Key outcomes from this objective include the development of improved statistical genetics abilities of PD Hershberger and the generation of knowledge that can be directly incorporated into sweet corn biofortification breeding programs. The results from this objective have been incorporated into a manuscript that will be submitted to a peer-reviewed journal during the next reporting period. Objective 3: Perform genomic prediction using information gained from the association studies Genomic prediction models incorporating transcriptome-based relationship matrices were also evaluated using the dataset generated in Objective 1 paired with existing data as described in Objective 2. A total of twelve models were compared, each incorporating a different relationship matrix or set of matrices. Tested matrices included a genomic relationship matrix using a previously published single nucleotide polymorphism marker dataset from the same diversity panel, transcriptome-derived relationship matrices from each of the three reference genome alignments (B73v4, PH297, and Ia453), a relationship matrix made up of only a priori candidate gene transcript abundances from the B73v4 alignment, and a matrix composed of the complementary set of all non-a priori gene transcript abundances from B73v4. Overall, prediction accuracies ranged from moderate to high, but a priori candidate gene-targeted prediction models underperformed compared to those using transcriptome-wide datasets.Consistent with previous studies examining other traits in maize, transcriptomic data increased prediction accuracies over genomic data alone for some but not all traits. Key outcomes from this objective include the development of an improved understanding of genomic selection and the development of reproducible analyses for PD Hershberger and the generation of knowledge that can be used to inform the optimal use of genomic selection methods in sweet corn sweet corn biofortification breeding programs. Along with the results from Objective 2, the results from this objective have been incorporated into a manuscript that will be submitted to a peer-reviewed journal during the next reporting period.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Hershberger JM, Baseggio M, Murray M, Magallanes-Lundback M, Kaczmar N, Wood JC, Chamness J, Buckler ES, Smith
ME, Buell CR, DellaPenna D, Tracy WF, and Gore MA. Integrating transcriptomics for the improvement of genetic
dissection and prediction of provitamin A and vitamin E in fresh sweet corn kernels. Poster presented at the 62nd Annual Maize Genetics Meeting; 2020 June 25-26; Virtual
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Progress 05/15/19 to 05/14/20
Outputs
Target Audience:Target audiences Cornell University Plant Breeding graduate students Cornell University undergraduate students Efforts Teaching assistant for Cornell University course PLBRG4030 (Genetic Improvement of Crop Plants) during fall semester 2019 including giving a lecture, providing office hours, and grading homework assignments and exams Teaching assistant for Cornell University course PLBRG4060 (Methods of Plant Breeding Laboratory) during fall semester 2019 including giving a lecture and creating and facilitating a laboratory activity on introductory programming and model building in R Guest lecture Cornell University course PLSCI4100 (Digital Technologies for Research and Communication) Development of introductory R programming tutorial and laboratory exercise for undergraduate and graduate students in the plant sciences Changes/Problems:Due to the COVID-19 pandemic, the Cornell campus was closed to all but essential research in mid-March 2020. Cornell defines essential research as "care for animals, plants and unique or expensive cell cultures or biological specimens, preservation of unique reagents and other unique or expensive materials, and maintaining equipment (e.g., liquid nitrogen and liquid helium systems, and shared computational clusters) that cannot be maintained remotely or shut down without significant cost or consequences to the research effort". Unfortunately, this project does not match Cornell's definition of essential. At the time of campus closure, PD Hershberger was not yet finished with RNA extractions for the tissue samples collected during the 2019 field season. The project cannot move forward in terms of data generation until campus reopens, but PD Hershberger is using the mandated time away from the laboratory to learn data analysis techniques for 3'RNA-seq data and to perform literature review. As soon as the Cornell campus reopens, PD Hershberger will resume RNA extractions as planned and the project will continue to move forward. For the time being, the samples are in the lab -80C freezer and are expected to retain integrity for a minimum of 12 months in their current state. In addition to direct research impacts, the COVID-19 pandemic led to the cancellation of the Maize Genetic Conference in March 2020. PD Hershberger prepared a poster to present the work of this project at this conference but was neither able to attend nor present the poster as planned due to the cancellation. The abstract is available for download from the meeting website. What opportunities for training and professional development has the project provided?Training activities In October 2019, PD Hershberger visited the laboratory of Dr. C. Robin Buell for one-on-one training on hot borate RNA extraction and 3'RNA-seq bioinformatics. Using the information learned from Dr. Buell's research group, PD Hershberger successfully replicated the RNA extraction method at Cornell University. The bioinformatics training will be put to use after the sequencing results have been generated. Three Cornell undergraduate students and two Ithaca High School students assisted PD Hershberger with field-related activities for this project from June - August 2019. These students were trained in the care and pollination of sweet corn plants as well as in phenotyping best practices. Professional development PD Hershberger planned to attend the Maize Genetics Conference in Kona, Hawaii in March 2020. An abstract related to this project was submitted and accepted by the conference selection committee, but unfortunately the conference was canceled due to concern over COVID-19 (https://www.maizegdb.org/docs/MGM2020-cancellation.pdf). How have the results been disseminated to communities of interest?The abstract submitted to the Maize Genetics Conference, while not presented in poster form, is available online to the maize genetics community (http://community.maizegdb.org/abstracts/mm2020/reports/see_updated_abstract.php?id=214&pin=XzqByC). What do you plan to do during the next reporting period to accomplish the goals?As soon as the Cornell campus reopens, PD Hershberger will continue RNA extractions. Following extractions, five 96-well plates will be submitted to the Cornell Genomics facility for library preparation and 3' RNA sequencing. These data will then be filtered, aligned, and analyzed according to the original proposal in order to accomplish the remaining project objectives. PD Hershberger will then write two manuscripts on the results of this project as described in the original project plan. In addition, PD Hershberger will present the results of this project at the National Association of Plant Breeders annual meeting in August 2020 and at the International Plant and Animal Genome conference in January 2021 if these conferences are not canceled due to the COVID-19 pandemic.
Impacts
What was accomplished under these goals?
Despite the importance of vitamin E and provitamin A for human health and well-being, dietary intake of these vitamins is suboptimal in the United States. Sweet corn, the third most highly consumed vegetable in the country, is amenable to breeding for increased nutritional quality, providing an opportunity to meet these dietary needs through biofortification. Through the sequencing and analysis of RNA from a diverse panel of sweet corn inbred lines, this project will provide a better understanding of the genetics underlying nutritional traits in fresh sweet corn kernels and the development of tools for plant breeders to apply this knowledge to the generation of superior plants. Though no results have yet been generated for this project, fresh kernel samples have been collected from the experimental sweet corn lines. RNA extraction is currently paused due to the closure of the Cornell University campus in response to COVID-19, but the experiment will continue as proposed as soon as the campus reopens. Objective 1: Collect novel gene expression data on a diverse panel of fresh sweet corn kernels Work is currently underway to complete this objective. A diverse panel of 380 sweet corn inbred lines was planted, grown, and pollinated at Cornell University's Musgrave Research Farm from May-August 2019. Including experimental checks, 440 samples of fresh kernels were harvested at 400 growing degree days after pollination during August and September 2019. After thorough training on RNA extraction with Dr. C. Robin Buell's research group at Michigan State University in October 2019, PD Hershberger submitted test samples of extracted RNA to the Cornell Genomics facility for quality control in December 2019. Library preparation and spike-in sequencing was successful for all of the submitted test samples. Frozen kernel tissue was ground in January and February 2020 and RNA extractions from experimental samples began in February 2020. Unfortunately, extractions were paused in early March 2020 due to the closing of the Cornell campus in response to COVID-19. Extractions will continue as soon as campus reopens. Key outcomes during this reporting period include improved skills for PD Hershberger related to experimental design and RNA extraction. Objectives 2 and 3 require the completion of objective 1 as input, so no concrete progress was made towards these objectives during this reporting period. However, PD Hershberger has been involved in informal training and literature review of the analyses outlined in these objectives in preparation for data availability during the next reporting period.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Please note that the following poster abstract was accepted but the conference was canceled due to COVID-19 (https://www.maizegdb.org/docs/MGM2020-cancellation.pdf), so the final poster was not presented:
Hershberger JM, Baseggio M, Murray M, Magallanes-Lundback M, Kaczmar N, Wood JC, Chamness J, Buckler ES, Smith ME, Buell CR, DellaPenna D, Tracy WF, and Gore MA. Integrating transcriptomics for the improvement of genetic dissection and prediction of provitamin A and vitamin E in fresh sweet corn kernels. Abstract accepted for the Maize Genetics Meeting; 2020 March 12-15; Kailua-Kona, HI
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