Progress 05/15/19 to 05/14/20
Outputs
Target Audience: ?Target audiencesinclude individuals, groups, market segments, or communities that will be served by the project. Where appropriate, you should also identify population groups such as racial and ethnic minorities and those who are socially, economically, or educationally disadvantaged. Effortsinclude acts or processes that deliver science-based knowledge to people through formal or informal educational programs. Examples include: formal classroom instruction, laboratory instruction, or practicum experiences; development of curriculum or innovative teaching methodologies; internships; workshops; experiential learning opportunities; extension and outreach. Formal Classroom Instruction 1.) Co-Instructor of Record for BIO_SCI:2150 Genetic Diseases (Online): included the development of assessment tools, dissemination of material, mentoring of students. Topics focused around human genetic diseases as a method to teach genetics to non-majors. Targetaudience:University of Missouri, upper-class non-majors, i.e. Junior and Senior level undergraduate students outside of Biology as a major. Included in my Graduate Minor in Collee Science Teaching outlined in my Training/Career Development Plan. 2.) Guest Lectures BIO_SCI:1010 General Biology (non-majors), "General Genetics" "Introduction to GMOs" "Pedigrees" "Evolution" Target audience:University of Missouri, required science course for non-major "science" credit, includes majority freshman non-majors, however all undergraduate grades are able to take this course. Included development and dissemination of information through lectures. BIO_SCI:3210 Plant Systematics (non-majors), "Monocot Evolution" Target audience:University of Missouri, upper-classman non-major science objective course, writing intensive. Included a lecture on overall monocot evolution,in which I was able to incorporate elements of my research, and a paper discussion over the development of the monocot phylogeny. BIO-125 Genetic Engineering (majors), "Crop domestication, breeding, and the future with genetic engineering" Target audience:Whitman College, Walla Walla Washington, upper-classman biology major elective course. This lecture included a hands on "mock" demonstration of traditional breeding versus breeding with genetic engineering tools as well as a brief lecture and Q&A. A part of my mentorship with Dr. Brit Moss and mock interview weekend outlined in my Training/Career Development Plan. Experiential Learning Opportunities 1.) Year-long mentoring experience for junior student majoring in Plant Sciences. This included mentoring him in writing a grant to fund his research (CAFNR Undergraduate Research Internship 2019-2020, Received, 2020-2021, Pending), and helping him direct that research over the Fall, Spring, and Summer semesters. This included training in SEM, confocal imaging, RNA extraction for hormone analysis, and double mutant analysis. In addition, research was presented at the 2020 Undergraduate Research Forum (online) in the form of a virtual poster. Target audience:University of Missouri, junior-level male, scio-economic minority student in Plant Sciences. This falls under the continue undergraduate mentoring outlined inTraining/Career Development. Workshops 1.) Equity in Syllabus Workshop Leader, THRIVE "Transform the Natural Sciences at Mizzou"Student Leader Presentation, HHMI Peer Implementation Cluster Meeting, University of Missouri - Columbia. Co-lead discussion on how to include equity best practices in course syllabus design with anundergraduate student,initiated critical analysis of current syllabi in four classes in the natural sciences and prompted subsequent discussion on successful equity approaches and areas to improve in syllabus design. Target audience:Student groups from three different universities, University of Missouri administration leadership, and undergraduate student leaders. This group focuses on serving the New Majority of students, including those historically under-represented in science (Black Americans, women of color, Latinx students, non-traditional students, students from low income-classes).This experience promotes Education and Workforce development via continuous training on how to teach and menotr New Majority students. Extension and Outreach 1.) HHMI THRIVE Program Mentor, graduate student mentor for small groups of undergraduate students working on projects to "Transform the Natural Sciences at Mizzou." The THRIVE program's main goal is to make a more inclusive environment in the natural sciences for New Majority students at the University of Missouri-Columbia. Target audience:This group focuses on serving the New Majority of students, including those historically under-represented in science (Black Americans, women of color, Latinx students, non-traditional students, students from low income-classes). This expereince promotes Education and Workforce development via continuous training on how to teach and menotr New Majority students. Changes/Problems:? Change in RNAseq experimental design Major problems in original design: the amount of tissue needed for one replicate required more plants than seed allowed in the growing season. In addition, efforts are still on-goingto accurately genotypeSos2plants before phenotypes emerge at reproductive maturity. Change in experimental design: I decided to collect tissue from whole immature tassels (3-5mm) for RNA extraction and RNAseq analysis. In addition, two other semi-dominantSosmutants have been identified:Sos1(Wu et al. 2009) andSos3which are also defective in the SPM to SMtransition, ultimately leading to paired spikelet production. In an effort to identify shared gene networks of these mutants responsible for the SPM to SMtransition in maize, whole immature tassels (3-5mm) were also extracted fromSos1andSos3mutants for analysis. This change still allows my to answer my major research objective:Understanding the role ofsos2in meristem networks required from proper growth and developmentwhile additionallybuilding a network of gene regulation during meristem transitions during developmentin a broader and more comprehensive way. Support for changes: SEM analysisfound SPM to SM transitions in both of these zones in the W22 background, confirming the accuracy in analyzing this transition in whole 3-5mm tassels, which allowed for larger tissue sampling for RNA extraction. Major activities completed: RNA extraction, library prep, and differential expression analysis of all threeSosmutants Data collected: transcriptome profiles ofSos1/+, Sos2/+, and Sos3/+mutant plants and associated WT siblings for comparison. Will be submitted to an open access platform upon submission of manuscript Summary of results: At this stage of development, between 61-68% of the genome is expressed. Of the total nonzero read counts collected from all threeSosmutants, a core of 13,376 genes were found to be shared between all.Sos2andSos3mutant transcriptomic profiles were the most alike withSos1being the most different, which correlates with the level of severity of the heterozygous phenotype collected (Sos2andSos3are more severe in phenotype thanSos1). When transcriptomic profiles were narrowed down to differentially expressed genes via DESeq2, similarities between mutants also dampened. A total of 368 DEGs were expressed inSos1mutants, 2.422 DEGs inSos2,and 479 DEGs inSos3, and only 5 of these genes overlapped between the three mutants.Sos2andSos3still shared more DEGs thanSos2andSos1orSos3andSos1,but these mutants have primarily unique DEGs. Further analysis of results will be reported in the second year reporting period. No other major changes have been made to the initial proposal. What opportunities for training and professional development has the project provided? Training activities Mentorship by Co-PIs Drs. Paula McSteen and Dr. Trupti Joshi via bi-weekly/monthly meetings. These meetings included discussion of research progress, mentorship in grant and paper writing, and figure design for future papers. In addition, Dr. McSteen aids in career development and navigating job market and career networking. Professional development Mock Interview Weekend, Whitman College, Walla Walla, Washington. Set up by mentor/collaboratorDr. Brit Moss, this weekend served to prepare me to enter the job market, specifically at as a faculty member at a PUI with a research focus. The first day included a series of interviews with faculty and students,a research presentation to faculty, and staff in the Biology department and a teaching demonstration (lecture/activity in Genetic Engineering, active class). The second day was observation of research based undergraduate laboratory course: Synthetic Cell Biology for potential application of knowledge in a future laboratory-research program. Inclusion, Diversity, Race Book club - University of Missouri Biological Sciences Graduate Program (ongoing), designed to help foster discussion on race, inclusion, and diversity in our department in order to be intentional in accepting black Americans, women of color, Latinx, and other historically underrepresented groups in our teaching, research, and future careers. How have the results been disseminated to communities of interest?? Dissemination of results The research results from this reporting periods work was intended to be disseminated at two conferences this year: ASPB Midwest Section Meeting, Urbana-Champaign, IL (acceptedoral presentation) - this was canceled due to COVID and has not be rescheduled 62nd Maize Genetics Conference, meeting in Kona, HI canceled due to COVID, virtual meeting commencedJune 25-26, 2020 (accepted poster presentation, presented virtually viaZoom) Education/Outreach to communities notusually aware of research: Southfarm Showcase (2019) - a community outreach event, specifically geared towards children (K-8), to teach them about the science that takes place at the Missouri Mazie Center. My role in this was to help kids work through questions about plant development. Science Night at Jefferson Farm and Garden (2019) - a community outreach event that allows graduate students (specifically those in plant research) the opportunity to present their research to the general public. I served as a panelist during the community questioning period where I was able to briefly describe my research (funded by this grant) and answer community questions about it. NOTE: The COVID pandemic has stalled many outreach and educational activities during the last few months of this reporting period, and continues to stall efforts as the next reporting period starts. I predict that this will impact next year's progress REEport as well - and am currently working on finding solutions to continue to disseminate my research to both the science and local non-science communities. What do you plan to do during the next reporting period to accomplish the goals?? The biggest difficulty this project faces looking forward to the next reporting period is the ongoing COVID pandemic. Currently, I have been granted permission to work both in the lab and at my associated research field site with the understanding that as much work I can do at home I will.I do not predict that the previous two-month closure of the University orMissouri, or the possible future closure of MU if the pandemic continues, will impact the progress of my research, but if it does, I will update my program manager with a detailed plan of action on how to address those barriers as they come. I will continue to carry out the research outlined as proposed and am confident that I will stay on track for successful completion next reporting period.
Impacts
What was accomplished under these goals?
Research Impact Grasses, such as maize (corn), rice, wheat, and barley, constitute major sources of nutrients necessary to feed the world. These grains are produced from spikelets, the short branches that produce flowers in grasses, that arise from a central group of stem cells called a meristem. A notable difference in spikelet development between grass species is that in wheat and rice spikelets are produced singly, while members of the Andropogoneae tribe, such as maize, produce spikelets in pairs essentially allowing them to double grain output. The goal of my research is to better understand the genes that control this development in order to help inform the improvement of grass crops in the future. I utilize theSos2maize mutant, defective in paired-spikelet development, to understand what genes a normally required for proper paired-spikelet production. I have discovered that at least two genes and the temperature of the growing environment affect theSos2phenotype. WhileSos2does affect known meristem regulatory pathways (supported by genetic and transcriptomic evidence outlined below), I also fund thatSos2transcripts are enriched for heat shock genes during tassel development. This is of compounding interest, asSos2is directly involved in the development of the structures responsible for plant yield, and changes expression of genes known to respond to temperature changes. The growing connection betweenSos2, a gene important in paired-spikelet development (and ultimately plant yield) and heat shock response (increasingly important to study in terms of plant growth andclimate change)is exciting and increases the potential impact of this research. ObjectiveProgress Objective 1: Cloning and verification of the suppressor of sessile spikelet2 (sos2) gene in maize. Major activities completed: Development of EMS M2 lines for analysis this summer (2020). This includes the EMS of W22-ACR pollen crossed to W22-ACR ears andSos2/+pollen crossed onto W22-ACR ears in 2018/2019, selfing of W22-ACR EMS M1 seed (i.e. creation of M2 seed to be analyzed in summer 2020)and analysis of Sos2/+ M1 lines for dominant alleles in 2019. Re-analysis of BSA whole genome sequencing data fromSos2plants (originally analyzed in 2018) Analysis of potential genetic and environmental modifiers of theSos2phenotype. Data Collected: 240Sos2EMS M1 seedsand 3,690 W22-ACR M1 seeds were analyzed for dominant Sos2 alleles Entire genome of Sos2 was re-analyzed for other potential candidate gene regions, outside of previously defined fine-mapping window on the short arm of chromosome 10. To determine if variation in phenotype was due to epistatic effects between two genes, 393 plants, segregating 1:2:1 were analyzed in MO2019. Intensive phenotypic data was collected from mature tassels and ears, and scored 1-6 in increasing severity, 1 being WT and 6 being not present. To determine if the variation of phenotype was due to environmental modifiers acting on a semi-dominant mutation, 96Sos21:1 segregating seeds were planted in growth chambers under two different environmental conditions ("hot" and "cool" average daily temperatures) until adulthood. reproductive phenotypes were scored the same way as described in #4, above. Summary of results: No additional allele of Sos2 found in 2019 analysis.Could be due to low seed set resulting in a statistically low chance of finding dominant EMS mutation Discovered two additional regions of the genome that could have potential impacts onSos2phenotype, either modifiers or causative genes. Statistically, phenotypicratios supported dominant-recessive epistatic gene interaction. TheSos2phenotype is suppressed in "hot" growth conditions (85F/29C day temp, 65F/18C night temp) compared to "cool" (75F/24C day temp, 65F/18C night temp) conditions, This data correlates with the results from the RNAseq analysis, described in Obj 2 below. Key outcomes: A valuable change in knowledge includes the understanding that a strong intersection between a causative gene, genetic modifiers, and environmental modifiers affect the development of the paired-spikelet in maize. This developmental trait is morenuancedthat previously thought, andresemblein research ofother multi-factorial, economically important developmental traits, such as plant height. Objective 2: Understanding the role of sos2 in meristem networks required from proper growth and development. Major activities completed: Double mutant analysis of Sos2 and known meristem mutants (fea2, td1, ct2). RNAseq analysis ofSos2immature tassels (modified from original proposal - seeDifficulties and Solutionsbelow and/orChanges/Problems). Preliminary conformation of transcriptomic results via RNA in situ(using the KN1 probe), and hormone measurements of Sos2 immature tassels. Dissemination of results at the 62nd Annual Maize Genetics Conference, held virtually June 25-26th, 2020. Data Collected: Phenotypic data collected fromSos2;fea2(140 total)andSos2;td1(232 total) segregatinglines grown in MO2019, andSos2;ct2(65 total)andSos2;fea2(45 total) segregating plants grown during the HI2020 field season. Transcriptomic data fromSos1, Sos2,andSos3immature tassels and associated WT siblings Hormone measurements onSos2immature tassels Summary of results: Sos2;ct2 andSos2;td1double mutants look similar toSos2reproductively, indicatingSos2is epistatic toct2andtd1in reproductive meristem development.Sos2;fea2double mutants look likeSos2in the tassel andfea2in the ear, indicatingSos2is epistatic tofea2in tassel development, but either does not function or functions downstream offea2during ear development. Sos2RNAseq analysis identified 2,789 differentially expressed genes vis EdgeR (FDR < 0.05) and 2,628 DEGs via DESeq2 (p < 0.05). There are roughly equal numbers of up and down regulated DEGs andGO analysisfound enriched GO terms involved in every stage of development (i.e. Leaf Development, Root Development, Flower Development). Additional enriched GO terms: Response to Heat, Response to Misfolded Protein. A significant increase of cytokinin intermediaries cis- and trans-zeatin (p < 0.001), and a significant decrease in ABA, IAA, four GA intermediaries (p < 0.05). A core 13,376 genes are expressed in all threeSosmutants, and Sos2/Sos3 gene expression profiles are most similar overall. Key outcomes: Research progress on this objective during this reporting period has led to the hypothesis that the gene responsible for the Sos2 is involved in all meristem types throughout development. While RNAseq supports a role of theSos2gene in meristem maintenance, it also indicatesthat heat shock proteins play some role, direct or indirect, inSos2meristem development. This finding also ties into the observation that the Sos2 tassel phenotype is suppressed at hot temperatures (discovered in Obj. 1).
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Guthrie, "Sequencing and genetic characterization analysis of the Suppressor of sessile spikelets (Sos) semi-dominant mutants in maize," ASPB Midwestern Section Meeting, Urbana-Champaign, IL, Oral Presentation (canceled due to COVID-19 pandemic)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Guthrie, Joshi, McSteen, "Genetic and transcriptomic analysis of the Suppressor or sessile spikelets2 (Sos2) mutant in maize," 2019, 62nd Virtual Maize Genetics Conference, Poster Presentation
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