Progress 10/01/16 to 09/30/21
Outputs
Target Audience:The primary target audience reached by our efforts during the reporting period has been other plant biologistsand evolutionary biologists. PI Blackman and other project participants have reached the target audience throughpublications and seminar or conference presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PI Blackman met weekly with all project scientists, postdoctoral associates, and graduate students associates involved with the project.Undergraduates met more than weekly with their mentors in the lab and several times a semester with PI Blackman. Atweekly lab meetings, project members presented research updates, received peer review of manuscript drafts, and discussedselections from the recent literature. PI Blackman has reviewed the faculty job application packages for twopostdoctoralassociates and one assistant project scientistaffiliated with the project. One undergraduate is completing their Honors Thesis based on their contributions to these projects, and 2 former undergraduate participants in this project co-authored publications published this year. During the reporting period, one former postdoctoral scientist associated with these projects started a research scientist position at Sound Agriculture, Inc. One graduate student passed their qualifying exam to advance to doctoral candidacy during the reporting period as well. How have the results been disseminated to communities of interest?The primary target audience reached by our efforts during the reporting period has been other plant biologists and evolutionary biologists. PI Blackman and other project participants have reached the target audience through publications and seminar or conference presentations. PI Blackman presented on his efforts related to this project in virtual departmental seminars at University of California, Berkeley, and University of California, Riverside and virtual contributed talks at the Evolution 2021 and Botany 2021 conferences. Assistant Project Scientist presented on his work on monkeyflower transformation virtually for the Plant Biology 2021 conference. Undergraduates Allison Rothrock and Aurora Gaspard delivered oral presentations about their projects to the Summer Undergraduate Research Fellowship Program and Amgen Scholars Program, respectively. We published one paper related to our work on the genomics of local adaptation in monkeyflower in July 2021 inMolecular Ecology.We also published one paper related to our work on sunflower reproductive biology in August 2021 inNew Phytologist. We also communicated our science to plant biotechnology industry groups, including an annual presentation to our gift sponsor, Innolea, and a talk by PI Blackman to the KWS Gateway Research Center. PI Blackman also communicated results from our work to the general public by speaking to the Yolo County Master Gardeners and spoke about his sunflower research briefly on NPR Weekend Edition Sunday. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Project Goal 1 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism in sunflower. Noprogress during the reporting period. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. Previously, we completed growth chamber experiments wheresunflower plants were being raised under 1) long days, 2) short days, or 3) long days followed by transfer to short days afterless than 2 weeks. Analysis of data from previously sequenciedRNA-Seq libraries for 24 samples was renewed and advanced by rotation student Lynne Hagelthorn during this reporting period. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism in sunflower. Noprogress during the reporting period. Project goal 4 - Genetic of natural variation in critcal photoperiod in Mimulus. During the reporting period, we advanced lines containing mutations inone of our critical photoperiod candidate genes using CRISPR/Cas9 methods we have developed in the lab as part of project 6, and we are working to characterize them.During this reporting period, we also wrote up and published population genomic analyses of resequencingdata from plants sampled from an elevation transect in the Cascades mountain range. Through these analyses, we identified genes for which allelic variation is distributed along environmental gradients, thus making them excellentcandidates for adaptation to climate.Noteworthy candidates may have roles in critical photoperiod adaptation (e.g., a homolog of GIGANTEA) and brassinosteroid regulation. Project Goal 5 - Flowering and fitness in native seasonal environments.We pursued another round of field experiments at high and low elevation with F4 individuals derived from our four high x low elevation crosses and attempted to scored flowering, fitness proxies, and other traits. However, a dry early season at the low elevation site led us to opt against planting germinants in at that site, and we will do this in spring 2022. We were able to complete a field season at the high elevation site. We continue to extract DNA from the large mapping panels we grew in the previous field season, and we will perform genotyping by sequencing on these samples over the next year for QTL mapping of these characters. Project Goal 6 - Methods development for functional genetics in sunflower and monkeyflower. We were previouslysuccessful in implementing and improving the protocol for transformation of the common monkeyflower, Mimulus guttatus, toexpress hairpin RNAs (hRNA) andperformtargeted knockouts to perturb the function of severalpigmentation genes. This year, we have continued extendingourgene editing efforts and moved forward transformation ofadditional constructs targeting pigmentation patterning, trichomeproduction, copper tolerance, or critical photoperiod candidate genes. Characterization of phenotypes for the transgenics related to trichome production and copper tolerance has revealed that these functional manipulations do influence the traits as predicted, thus confirming successful identification of genes responsible for adaptive phenotypic variation in wild monkeyflower populations. Supported by gift funding from a plant biotechnology company, a researchassociate in UC Berkeley's new Innovative Genomics Institute plant transformation facility was hired in March 2018 and sincethen has been working to develop and troubleshoot methods for sunflower regeneration and transformation using split seedand immature embryos. New postdoc Ryan Nasti has also been working to translate methods for de novo initiation of meristems from somatic tissue to sunflower as another approach for transgenic production.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Colicchio JM, Hamm LN, Verdonk HE, Kooyers NJ, Blackman BK. Adaptive and non-adaptive causes of heterogeneity in genetic differentiation across the Mimulus guttatus genome. Molecular Ecology 30: 6486-6507 (2021).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Creux NM, Brown EA, Garner AG, Saeed SC, Scher CL, Holalu SV, Yang D, Blackman BK, and Harmer SL. Flower orientation influences floral temperature, pollinator visits, and plant fitness. New Phytologist 232: 868-879 (2021).
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The primary target audience reached by our efforts during the reporting period has been other plant biologists and evolutionary biologists. PI Blackman and other project participants have reached the target audience through publications and seminar or conference presentations. PI Blackman presented on his efforts related to this project at University of Rochester, Cornell University, and the Argentine Genetics Congress. PI Blackman also gave a UC Berkeley College of Natural Resources Homecoming Lecture on his work in sunflower in October 2019. Undergraduate Yuju Shin delivered an oral presentation about her Honors Thesis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PI Blackman met weekly with all postdoctoral associates and graduate students associates involved with the project.Undergraduates met more than weekly with their mentors in the lab and several times a semester with PI Blackman. Atweekly lab meetings, project members presented research updates, received peer review of manuscript drafts, and discussedselections from the recent literature. PI Blackman has reviewed the faculty job application packages for onepostdoctoralassociateaffiliated with the project. One undergraduate completed their Honors Thesis based on their contributions to these projects, and 1 former high school teacher and 5 former undergraduate participants in this project co-authoredpublications published or accepted for publication this year. How have the results been disseminated to communities of interest?We published one papers related to our work on pigmentation in monkeyflower. One paper involving our work on natural variation in evolutionary responses to drought was accepted in August 2020 and will be published in the next reporting period. In addition,our recently published work on monkeyflower pigmentation reached a broader scientific audience and the general public by beingfeatured in news articles in theDaily Californian,National Geographic, andScience. What do you plan to do during the next reporting period to accomplish the goals?Project Goal 1- Genetic basis of transitions in photoperiodic flowering in wild sunflower. In the next reporting period,additional gene expression analysis of candidate genesand confirmation of QTLswith select genotyping will be completed. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. In the next reporting period,computational analyses of transcriptomic data obtained will be completed. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism insunflower. Tissue will be ground and protocols for carbohydrate assays will be tested in the next reporting period. Project goal 4 - Genetic of natural variation in critcal photoperiod in Mimulus. We will continue writing manuscripts involvingthe characterization of QTLs and candidate genes; continue characterizing candidate gene knockout lines; and performresequencing for additional Mimulus populations throughout California. Project Goal 5 - Flowering and fitness in native seasonal environments. In the next reporting period, analysis of transcriptomic data of tissue collected in the 2017 field seasonwill continue. We will also perform another season ofcommon-garden field studies at high and low elevation in CA for genetic mapping, phenotypic selection, and gene expressionanalysis of F4 families, and we will perform genotyping by sequencing and QTL analysis of the populations grown in the past field season. Project Goal 6 - Methods development for functional genetics in sunflower and monkeyflower. Following on our successesin expressing hRNAs to knockdown gene expression, using GUS reporter constructs to reveal spatial expression domains,effectively using CRISPR/Cas9 gene editing methods to produce targeted (multi-)gene knockouts in monkeyflower, we will continue expandingour efforts to apply our methods to examine the functions of additional genes and also extend our gene editing methods. We will continue to pursue increasing the efficiency and reducing the genotypespecificity of sunflower transformation using tissue culture protocols involving split seeds and immature embryos. We will also attempt to implement a new method for somatic meristem induction and transformation in both systems to avoid several steps of tissue culture and speed the transformation process.
Impacts
What was accomplished under these goals?
Project Goal 1- Genetic basis of transitions in photoperiodic flowering in wild sunflower. Previously, QTLs for flower weremapped in F3s from a day neutral (MB) x short-day (KS) cross and F2s from a short-day (KS) by long-day (TX) cross thatwere phenotyped under short days andlong days, and genotyped by double-digest genotyping-bysequencing. In both panels, we found several QTLs that arecommon to both short days and long days as well as other QTLs that are detected only in one photoperiod. The latter set ofQTLs are thus the regions most likely involved in transitions between the photoperiod response types (as opposed tocontrolling generally earlier or later flowering). This year, we have continued characterizingselected panels of candidategenes to examine for sequence and expression differences between the cross parents under short and long dayconditions. All RNA extractions are complete,and qRT-PCRis ongoing. We have obtained genotype information for these QTLs in a set offull sibs from the MBxKSpanel in the field at the Oxford Tract Facility in summer 2018 to quantify the impact of the QTL allelic variation on flowering, plantarchitecture, and fitness under seasonal conditions. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. Previously, we completed growth chamber experiments wheresunflower plants were being raised under 1) long days, 2) short days, or 3) long days followed by transfer to short days afterless than 2 weeks. Last year, we finished buildingRNA-Seq libraries for 24 samples and an undergraduate honors student dida preliminary analysis of this data. No additional progress was made this year, but a rotation student starting in winter 2020 will return to it. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism in sunflower. Noprogress during the reporting period. Project goal 4 - Genetic of natural variation in critcal photoperiod in Mimulus. During the reporting period, we generated a targeted knockout inone of our critical photoperiod candidate genes using CRISPR/Cas9 methods we have developed in the lab as part of project 6, and we are working to characterize it.We also published one paper related to our work on this topic describing our initial assessment of how phenotypic evolution during the 2010s Western US drought among Californian and Oregonian populations. Another key effort in the lab during this reporting period has population genomic analysis of resequencingadditional samples from an elevation transect in the Cascades mountain range. Through these analyses, we have identified genes for which allelic variation is distributed along environmental gradients, thus making them excellentcandidates for adaptation to climate.Noteworthy candidates may have roles in critical photoperiod adaptation (e.g., a homolog of GIGANTEA) and brassinosteroid regulation. Project Goal 5 - Flowering and fitness in native seasonal environments.We conducted another round of field experiments at high and low elevation with F4 individuals derived from our four high x low elevation crosses and attempted to scored flowering, fitness proxies, and other traits. However, due to pandemic constraints and atypical, challenging field conditions (dry, late season at low elevation; exceptionally high herbivory at high elevation), these experiments were a wash. We are currently extracting DNA from the large mapping panels we grew in the previous field season, and we will perform genotyping by sequencing on these samples over the next year for QTL mapping of these characters.In addition, RNAseq libraries were built ad sequenced from diurnal times series tissue collections from mapping panel parent lines grown in 2017 at high and low elevation in Oregon. Project Goal 6 - Methods development for functional genetics in sunflower and monkeyflower. We were previouslysuccessful in implementing and improving the protocol for transformation of the common monkeyflower, Mimulus guttatus, toexpress hairpin RNAs (hRNA) andperformtargeted knockouts to perturb the function of severalpigmentation genes. This year, we have continued extendingourgene editing efforts and moved forward transformation ofadditional constructs targeting pigmentation patterning, trichomeproduction, copper tolerance, or critical photoperiod candidate genes. One noteworthy advance is that we have been able to implement targeted knock out of multiple genes with one construct where multiple guide RNAs are concatenated by tRNA linkers. We also published our first paper including RNAi and CRISPR edited plants generated by this work. Supported by gift funding from a plant biotechnology company, a researchassociate in UC Berkeley's new Innovative Genomics Institute plant transformation facility was hired in March 2018 and sincethen has been working to develop and troubleshoot methods for sunflower regeneration and transformation using split seedand immature embryos.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ding B&, Patterson EL&, Holalu SV&, Li J, Johnson GA, Stanley LE, Greenlee AB, Peng F, Bradshaw HD, Blinov ML, Blackman BK*, Yuan Y-W*. Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns. Current Biology 30: 802-814 (2020). (&equal contributions, *co-senior authors)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Kooyers NJ, Morioka KA, Colicchio JM, Clark KS, Donofrio A, Estill SK, Pascualy CR, Anderson IC*, Hagler M, Cho C, Blackman BK. Population responses to a historic drought across the range of the common monkeyflower (Mimulus guttatus). American Journal of Botany
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The primary target audience reached by our efforts during the reporting period has been other plant biologists and evolutionary biologists. PI Blackman and other project participants have reached the target audience through publications and seminar or conference presentations. PI Blackman also delivered the one of the two 2019 UCB College of Natural Resources Homecoming Lectures, bringing some of these efforts to a public audience. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PI Blackman met weekly with all postdoctoral associates and senior research associates involved with the project.Undergraduates met more than weekly with their mentors in the lab and several times a semester with PI Blackman. Atweekly lab meetings, project members presented research updates, received peer review of manuscript drafts, and discussedselections from the recent literature. PI Blackman has reviewed the faculty job application packages for onepostdoctoralassociateaffiliated with the project. Two undergraduates completed Honors Theses based on their contributions to these projects. How have the results been disseminated to communities of interest?The primary target audience reached by our efforts during the reporting period has been other plant biologists and evolutionary biologists. PI Blackman and other project participants have reached the target audience through publications and seminar or conference presentations. PI Blackman presented on his efforts related to this project at the International Consortium for Sunflower Genomics annual meeting, Mimulus Meeting 2019, Plant Biology 2019, andCalifornia State University, Chico.Postdoctoral fellow Srinidhi Holalu presented on his work performing functional studies of pigmentation patterning in monkeyflower at the Plant Biology 2019 meeting. Undergraduates Adrian Overly and Brittnie Phan madeposter presentations about their Honors Theses. We published one paper related to our work on monkeyflower, and we published an invitedreviewpaper specifically on the topic of this project: the genetics of flowering time evolution. One paper involving our work on pigmentation in monkeyflower which was previously submitted to bioRxiv is now in press and will be published in the next reporting period. What do you plan to do during the next reporting period to accomplish the goals?Project Goal 1- Genetic basis of transitions in photoperiodic flowering in wild sunflower. In the next reporting period,additional gene expression analysis of candidate genesand confirmation of QTLswith select genotyping will be completed. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. In the next reporting period, computational analyses of transcriptomic data obtained will be completed. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism insunflower. Tissue will be ground and protocols for carbohydrate assays will be tested in the next reporting period.Project goal 4 - Genetic of natural variation in critcal photoperiod in Mimulus. We will continue writing manuscripts involvingthe characterization of QTLs and candidate genes; genotyping F4 recombinants and phenotyping their progeny; perform another season ofcommon-garden field studies at high and low elevation in CA for genetic mapping, phenotypic selection, and gene expressionanalysis of F4 families; perform genotyping by sequencing and QTL analysis of the populations grown in the past field season,finishpopulation genomics analysis of populations along oneelevationcline. Project Goal 5 - Flowering and fitness in native seasonal environments. In the next reporting period, transcriptomicexamination of tissue collected in the 2017 field seasonwill continue. Project Goal 6 - Methods development for functional genetics in sunflower and monkeyflower. Following on our successesin expressing hRNAs to knockdown gene expression, using GUS reporter constructs to reveal spatial expression domains,and effectively using CRISPR/Cas9 gene editing methods to produce targeted gene knockouts in monkeyflower, we will continue expandingour efforts to extend our gene editing methods. We are trying to obtain multi-knockout plants with constructscapable of expressing 8 guide RNAs (targets are pigmentation and trichome formation candidate genes) and targeting someinitial photoperiodic flowering candidate genes. We will continue pursue increasing the efficiency and reducing the genotypespecificity of sunflower transformation using tissue culture protocols involving split seeds and immature embryos.
Impacts
What was accomplished under these goals?
Project Goal 1- Genetic basis of transitions in photoperiodic flowering in wild sunflower. Previously, QTLs for flower weremapped in F3s from a day neutral (MB) x short-day (KS) cross and F2s from a short-day (KS) by long-day (TX) cross thatwere phenotyped under short days andlong days, and genotyped by double-digest genotyping-bysequencing. In both panels, we found several QTLs that arecommon to both short days and long days as well as other QTLs that are detected only in one photoperiod. The latter set ofQTLs are thus the regions most likely involved in transitions between the photoperiod response types (as opposed tocontrolling generally earlier or later flowering). This year, we have continued characterizingselected panels of candidategenes to examine for sequence and expression differences between the cross parents under short and long dayconditions. All RNA extractions are complete,and qRT-PCRis ongoing. We have also been obtaining genotype information for these QTLs in a set offull sibs from the MBxKSpanel in the field at the Oxford Tract Facility in summer 2018 to quantify the impact of the QTL allelic variation on flowering, plantarchitecture, and fitness under seasonal conditions. Quantification of seed counts as a proxy for fitness from this experiment was also completed during this period. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. Previously, we completed growth chamber experiments wheresunflower plants were being raised under 1) long days, 2) short days, or 3) long days followed by transfer to short days afterless than 2 weeks. This year, we finished buildingRNA-Seq libraries for 24 samples of leaf and meristem tissue collected from these plants and processed for gene theexpression studies. An undergraduate honors student dida preliminary analysis of this data, but we will do a more thorough analysis this year. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism in sunflower. Noprogress during the reporting period. Project goal 4 - Genetic of natural variation in critcal photoperiod in Mimulus. During the reporting period, weadvanced germplasm resources for fine mapping and transcriptomic analyses. Wecontinued to genotype three of the panels to identify panels segregating for recombination events in QTL intervals.We also worked toward generating a targeted knockout inone of our critical photoperiod candidate genes using CRISPR/Cas9 methods we have developed in the lab as part of project 6.During this reporting period, we have published one paper related to our work on this topic. We also been finalizing experiments and analyses for additional publications of ourdata on this topic. Another key effort in the lab during this reporting period has beenresequencingadditional samples from an elevation transect in the Cascades mountain range. All sequencing is complete and population genomic analyses are underway.Doing so willalow us to identify genes for which allelic variation is distributed along environmental gradients, thus making them excellentcandidates for adaptation to climate. Project Goal 5 - Flowering and fitness in native seasonal environments.We conducted field experiments at high and low elevation with F4 individuals derived from our four high x low elevation crosses and scored flowering, fitness proxies, and other traits. We are currently extracting DNA from these large mapping panels, and we will perform genotyping by sequencing on these samples over the next year for QTL mapping of these characters. Project Goal 6 - Methods development for functional genetics in sunflower and monkeyflower. We were previouslysuccessful in implementing and improving the protocol for transformation of the common monkeyflower, Mimulus guttatus, toexpress hairpin RNAs (hRNA) andperformtargeted knockouts to perturb the function of severalpigmentation genes. This year, we have been extendingourgene editing efforts and moved forward transformation ofadditional constructs targeting pigmentation patterning, trichomeproduction, or critical photoperiod candidate genes. Supported by gift funding from a plant biotechnology company, a researchassociate in UC Berkeley's new Innovative Genomics Institute plant transformation facility was hired in March 2018 and sincethen has been working to develop and troubleshoot methods for sunflower regeneration and transformation using split seedand immature embryos.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Lowry DB, Sobel JM, Angert AL, Ashman T-L, Baker RL, Blackman BK, Brandvain Y Byers KJRP, Cooley AM, Coughlan JM, Dudash MR, Fenster CB, Ferris KG, Fishman L, Friedman J, Grossenbacher DL, Holeski LM, Ivey CT, Kay KM, Koelling VA, Kooyers NJ#, Murran CJ, Muir CD, Nelson TC, Peterson ML, Puzey JR, Rotter MC, Yuan Y-W, Seemann JR, Sexton JP, Sheth SN, Streisfeld MA, Sweigart AL, Twyford JP, Vallejo-Mar�n M, Wu CA, Willis JH, Yuan Y-W. The case for the continued use of the genus name Mimulus for all monkeyflowers. Taxon 68: 617-623.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Gaudinier A and Blackman BK. Evolutionary processes from the perspective of flowering time diversity. New Phytologist 225: 1883-1898.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The primary target audience reached by our efforts during the reporting period has been other plant biologists andevolutionary biologists. PI Blackman and other project participants have reached the target audience through publicationsand seminar or conference presentations. Changes/Problems:For Project Goals 4 and 5, we have had challenges generating near isogenic lines for our QTL regions due to pest issues and inbreeding depression. We are altering our plans for what germplasm to use in expression and field studies to characterize the effects of variation at these loci on the flowering network, flowering, and fitnessin controlled conditions and the field. What opportunities for training and professional development has the project provided?PI Blackman met weekly with all postdoctoral associates and senior research associatesinvolved with the project. Undergraduates met more than weeklywith their mentors in the lab and several times a semester with PI Blackman. At weekly lab meetings, project members presented research updates, received peer review of manuscript drafts, and discussed selections from the recent literature. PI Blackman has reviewed thefaculty job application packages for four postdoctoral associates affiliated with the project. Dr. Nicholas Kooyers received feedback from PI Blackman and the lab on his research seminar prior to going for interviews, and he recently joined the faculty at the University of Louisiana, Lafayette.PI Blackman also attended presentations for junior faculty in the College of Natural Resources by Michel Estefan of the campus GSI Teaching and Resource Center on micro-aggressions and byKaren Nielsen of the campus Disabled Students Program on teaching and accommodating students with disabilities in the classroom. How have the results been disseminated to communities of interest?The primary target audience reached by our efforts during the reporting period has been other plant biologists and evolutionary biologists. PI Blackman and other project participants have reached the target audience through publications and seminar or conference presentations. PI Blackman presented on his efforts related to this project at the USDA ARS Plant Gene Expression Center; Understanding Natural Variation to Educate, Innovate, and Lead (UNVEIL) Research Coordination Network Symopsium on the Evolutionary Genomics of Adaptation; the "ASTA Meets UC Berkeley" meeting at the Innovative Genomics Institute (IGI) at UC Berkeley; the IGI Agricultural Reviews; and during his Botanical Society of America Emerging Leader Special Lecture at the Botany 2018 Meeting. Postdoctoral fellow Nicholas Kooyers presented on his related research efforts at University of Louisiana, Lafayette, Tulane University, Mississippi State University, University of South Carolina, Auburn University, and at the 2ndJoint Congress on Evolutionary Biology. Undergraduates Adrian Overly and Isaac Diaz made final presentations to their peers and mentors in their respective summer research internship programs. We have one paper in press at American Naturalistderiving from our work on the genetics of natural variation in monkeyflower. Another paper from this project and one involving our efforts to test gene function through plant transformation in monkeyflowerhave been submitted for review during the reporting period have also been posted as pre-prints on bioRxiv. PI Blackman,Dr. Kooyers, Dr. Colicchio, and undergraudate Shayne Estillhave also been active in preparing and launching a pilot citizen science program for phenological monitoringof high elevation plant taxa along the Pacific Crest Trail. We have worked with staff at two national parks and one national forest to obtainpermits and to identify sites and plants within sites for monitoring, and with our collaborators at the USA National PhenologyNetwork, wehave further developour website for the program through posting of training videos and site/phenophase guides (pct.usanpn.org). What do you plan to do during the next reporting period to accomplish the goals?Project Goal 1- Genetic basis of transitions in photoperiodic flowering in wild sunflower. In the next reporting period, quantification of seed counts as a proxy for fitness,geneexpression analysis of candidate genes, and confirmation of QTLs with select genotyping will be completed. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. In the next reporting period,RNA-Seq library sequencing andcomputational analyses will be completed. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism in sunflower.Tissue will be ground and protocols for carbohydrate assays will be tested in the next reporting period. Project goal 4 - Genetic of natural variation in critcal photoperiod in Mimulus. We will continuewriting manuscripts involving the characterization of QTLs and candidate genes; genotyping F4 recombinants and phenotyping theirprogeny; being common-garden field studies at high and low elevation in CA forgenetic mapping, phenotypic selection, and gene expression analysis of F4 families;finishshotgun sequencing and begin population genomics analysis of populations along twoelevation clines. Project Goal 5 - Flowering and fitness in native seasonal environments. In the next reporting period, transcriptomic examination of tissue collected in the 2017 field season and construction of NILs will continue. Project Goal 6 - Methods development for functional genetics in sunflower and monkeyflower. Following on our successes inexpressing hRNAs to knockdown gene expression, usingGUS reporter constructs to reveal spatial expression domains, andeffectively using CRISPR/Cas9 gene editing methods to produce targeted gene knockoutsin monkeyflower, we are ramping up our efforts to extend our gene editing methods. We are trying to obtain multi-knockout plants with constructs capable of expressing 8 guide RNAs (targets are pigmentation and trichome formation candidate genes) and targeting some initial photoperiodic flowering candidate genes. We will continue pursue increasing the efficiency and reducing the genotype specificity of sunflower transformation usingtissue culture protocols involving split seeds and immature embryos.
Impacts
What was accomplished under these goals?
Project Goal 1- Genetic basis of transitions in photoperiodic flowering in wild sunflower. Previously, QTLs for flower were mapped inF3s from a day neutral(MB) x short-day (KS) cross and F2s from a short-day (KS) by long-day (TX) cross that were phenotyped under short days and long days, and genotyped bydouble-digest genotyping-bysequencing. In both panels, we foundseveral QTLs that are common to both short days and long days as well as other QTLs that are detected only in onephotoperiod. The latter set of QTLs are thus the regions most likely involved in transitions between the photoperiod responsetypes (as opposed to controlling generally earlier or later flowering). Because the sequencing depth we chose left us withample missing data, we have been genotyping markers (either pre-developed or developed from SNPs in this dataset) to fill inthose gaps and to allow for better point estimates of GxE and GxG QTL effects. We have also reviewed the homology ofgenes that fall within our QTL intervals to Arabidopsis genes, and based on this knowledge we selected panels of candidate genes to examine for sequence and expression differences between the cross parents under short and long day conditions.We are currently extracting RNA from these collections for qRT-PCR. We also grew full sibs from the MBxKS panel in the field at the Oxford Tract Facility this summer to quantify the impact of the QTL allelic variation on flowering, plant architecture, and fitness under seasonal conditions. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. We have completedgrowth chamber experimentswhere sunflower plants were being raised under 1) long days, 2) short days, or 3) long days followed by transfer to short daysafter less than 2 weeks. Leaf and meristem tissue has been collected from these plants and processed for gene expressionstudies. Based on qRT-PCR examination of key reporters of floral induction, we selected 24 samples and made RNA-Seq libraries from them, and these libraries have been submitted for sequencing. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering and carbohybrate metabolism in sunflower.No progress during the reporting period. Project goal 4 - Genetic of natural variation in critcal photoperiod in Mimulus. During the reporting period, we advancedgermplasm resourcesfor fine mapping and transcriptomic analyses. Specifically, we finished collections of selfed seed for progeny testingand leaftissue forgenotyping from four four high x low elevationcrosses to the F4 generation. We continue togenotypethree of the panels to identify panelssegregating for recombination events in QTL intervals.For the one panel where genotyping has beencompleted, wehave performedprogenytesting for fine mapping. NIL construction has hit a major roadblock as most of our lines are showing low seed production due to exposure ofinbreeding depression after several generations of backcrossing.Therefore, we are moving forward to use our F4 panels or develop geneedited germplasmfor planned expression and field experiments.During this reporting period, we have published one paper and sent anotherpaper for review related to our work on this topic. Wealso been finalizing experiments and analyses for additionalpublications of our data onthis topic. In one of thesepublications, we will report the QTL analysis of one cross; geneexpression studies revealing that cis-regulatorydifferences affecting transcription of a tandem array of major floweringrepressors is likely the primary cause underlying one of these QTLs;field studies showing that two of the QTLs for criticalphotoperiod have significant effects on flowering and/or fitness in high and low elevationnative habitats; and populationgenomics studies that reveal signatures of selective sweeps coincident with the critical photoperiod QTLregions.With respect to the last finding, another key effort in the lab during this reporting period has been the successful adoption of a protocol for production of barcoded, shotgun sequencing libraries using microvolume amounts of the Illumina Nextera kit,essentially allowing us to produce 20x more libraries from this expensive commercial product. We used this method togenerate the 40 individual genomic libraries contributing to the manuscript in preparation, and haveuse this methodto re-sequence additional samples from quite a few more populations total sampled along two elevation gradients in the Cascades andSierra Nevada mountain ranges. We are now constructing the final libraries for one year of our sampling.Doing so will alow us to identify genes for which allelic variation is distributed alongenvironmental gradients,thus making them excellent candidates for adaptation to climate. However, wehave made these samecollections for six seasons now(including the one in the current period) to support future work examining how allelefrequencies shift across time (including from thebeginning to the end of the recent multi-year drought in the western US). In addition to support such future work, this summer we grew,generated ample outcrossed seed, and lightly phenotyped a refresher generation for >50 populationsfrom seed collections made in 2011,2016, and 2017. Project Goal 5 - Flowering and fitness in native seasonal environments. Germplasm progression was moved forward asdetailed forthe previous project goal. Project Goal 6 - Methods development for functional genetics in sunflower and monkeyflower. We werepreviously successfulin implementingandimprovingthe protocol for transformation of the common monkeyflower, Mimulus guttatus,to express hairpin RNAs (hRNA) targeting several genes affecting nectar guide pigmentation patterning and show the spatial expression pattern driven by the promoter of one of these genes using aGUS reporter fusion construct. Building on this success, we have successfully designed guide RNAs and used a construct carrying them and Cas9 to knockout function of a pigmentation patterning gene. We are the first group to do so for any species in the genus Mimulus.We are now expaning our gene editing efforts and are in the process of transforming additional constructs targeting pigmentation patterning, trichome production, or critical photoperiod candidate genes.Supported bygift funding from a plant biotechnology company, a research associatein UC Berkeley's new Innovative Genomics Institute plant transformation facility was hired in March 2018 and since then has been working to develop and troubleshoot methods for sunflower regeneration and transformation using split seed and immature embryos.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2018
Citation:
Kooyers NJ, Colicchio JM, Greenlee AB, Patterson EL, Handloser NT, Blackman BK. Lagging adaptation to climate change supersedes local adaptation to herbivory in an annual monkeyflower. American Naturalist (in press).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Ding B, Patterson EL, Holalu SV, Li J, Johnson GA, Stanley LE, Greenlee AB, Peng F, Bradshaw HD, Blackman BK, Yuan Y-W. Formation of periodic pigment spots by the reaction-diffusion mechanism.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Flagel L, Blackman BK, Fishman L, Monnahan PJ, Sweigart A, Kelly JK. GOOGA: A platform to synthesize mapping experiments and identify genomic structural diversity. PLoS Computational Biology.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The primary target audience reached by our efforts during the reporting period has been other plant biologists and evolutionary biologists. PI Blackman and other project participants have reached the target audience through publications and seminar or conference presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided??PI Blackman met weekly with all postdoctoral associates, graduate students, and senior research associates involved with the project. Undergraduates met more than weekly with their mentors in the lab and several times a semester with PI Blackman. At weekly lab meetings, project members presented research updates, received peer review of manuscript drafts, and discussed selections from the recent literature. Postdoctoral associate Jack Colicchio successfully applied for an NIH NRSA fellowship, and PI Blackman gave him several rounds of comments as he prepared his application. PI Blackman has reviewed the faculty job application packages for three postdoctoral associates affiliated with the project, and Dr. Nicholas Kooyers received feedback from PI Blackman and the lab on his research seminar prior to going for interviews. Postdoctoral associate Jack Colicchio applied for and received a travel award to the 40th New Phytologist Symposium "Plant epigenetics: from mechanisms to ecological relevance." He and postdoctoral associate Nicholas Kooyers also attended and presented at Evolution 2017. Postdoctoral associate Srinidhi Holalu presented at the International Plant and Animal Genome Conference XXV. PI Blackman attended the "Nature Research: Health of Research Groups" Symposium at UC Berkeley. PI Blackman also attended a presentation for junior faculty in the College of Natural Resources by Dr. Linda Von Hoene, of the campus GSI Teaching Resource Center, on the subject of faculty mentoring of graduate students. How have the results been disseminated to communities of interest?PI Blackman wrote a review about the genetics of natural variation in the plasticity of flowering time for Plant Physiology.PI Blackman presented on his efforts related to this project the International Plant and Animal Genome Conference XXV, University of San Francisco, UC Santa Barbara, UC Davis, the International Cluster of Excellence on Plant Science Summer School, the Max Planck Institute for Plant Breeding Research, Mimulus Meeting 2017, the Ward Watt Festschrift at Rocky Mountain Biological Laboratory, San Francisco State University, CSU Fresno, and Willamette University. Postdoctoral fellow Nicholas Kooyers presented on his related research efforts at Purdue University, NC State University, and Georgia Southern University. Postdoctoral fellow Srinidhi Holalu presented on his research at the project the International Plant and Animal Genome Conference XXV and the UC Berkeley Department of Plant and Microbial Biology retreat. PI Blackman and Dr. Kooyers have also been active in preparing a pilot citizen science program for phenological monitoring of high elevation plant taxa along the Pacific Crest Trail. We have worked with park staff at two national parks to obtain permits and to identify sites and plants within sites for monitoring, and our collaborators at the USA National Phenology Network have developed a website for the program (pct.usanpn.org). What do you plan to do during the next reporting period to accomplish the goals?Project Goal 1- Genetic basis oftransitions in photoperiodic flowering in wild sunflower. In the next reporting period, gene expression analysis of candidate genes and confirmation of QTL with select genotyping will be completed. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. In the next reporting period, RNA-Seq libraries will be constructed and sequencedfrom the RNAs we have extracted, and computational analyses wil be initiated. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering andcarbohybrate metabolism insunflower. Tissue will be ground and protocols for carbohydrate assays will be tested in the next reporting period. Project goal 4 -Genetic of natural variation in critcal photoperiod in Mimulus. We are in the process of writing several manuscripts involving the characterization of QTLs and candidate genes; genotyping F4 recombinants and phenotyping their progeny; and construction of NILs; and conducting shotgun sequencing forpopulation genomics of populations along two elevation clines. These efforts will continue throughthe next reporting period. Project Goal 5 - Flowering and fitness in native seasonal environments. In the next reporting period, transcriptomic examination of tissue collected in the 2017 field season and construction of NILs will continue. Project Goal6 -Methods development for functional geneticsin sunflower and monkeyflower.Following on our successes in transforming monkeyflower with constructs expressing hRNAs to knockdown gene expression or GUS reporter constructs, we will continue to pursue implementation of CRISPR/Cas9 gene editing methods in this species. Initial efforts will focus on pigmentation phenotypes but we also ancitipate pursuing constructs aimed at candidate genes for photoperiodic flowering. Supported by ouracquisition of funds to support a research associate focused on sunflowerat the Innovative Genomics Institute plant transformation facility, we will pursue increasing the efficiency and reducing the genotype specificity of the current sunflower transformation through tissue culture protocol, and we will also pursue other methods that may be more suited for DNA-free CRISPR/Cas9 gene editing methods.
Impacts
What was accomplished under these goals?
Project Goal 1- Genetic basis oftransitions in photoperiodic flowering in wild sunflower. Previously, F3s from a dayneutral (MB) x short-day (KS) cross and F2s from a short-day (KS) by long-day (TX) cross were phenotypedunder short days and long days, and DNAs were extracted from these individuals as well. We have now completed double-digest genotyping-by-sequencing (ddGBS) library constrution for these mapping panels and analyzed the resulting data. In both panels, we find several QTLs that are common to both short days and long days as well as other QTLs that are detected only in one photoperiod. The latter set of QTLs are thus the regions most likely involved in transitions between the photoperiod response types (as opposed to controlling generally earlier or later flowering). Because the sequencing depth we chose left us with ample missing data, we are now genotyping markers (either pre-developed or developed from SNPs in this dataset) to fill in those gaps and to allow for better point estimates of GxE and GxG QTL effects. We have also reviewed the homology of genes that fall within our QTL intervals to Arabidopsis genes,and based on this knowledge weselected panels of candidate genes to examine for sequence and expression differences between the cross parents under short and long day conditions. To facilitate the gene expression work, we have grown the parent accessions and collected tissue from them over a diurnal time course under long days, and we are currently extractingRNA from these collections. AnequivalentRNA resourceis already available for short days fromPI Blackman's previous studies. Project Goal 2 - Transcriptomics of LD->SD response in sunflower. We are currentlycompletinggrowth chamber experiments where sunflower plants are being raised under 1) long days, 2) short days, or 3) long days followed by transfer to short days after less than 2 weeks. Leaf and meristem tissue will be collected from these plantsand then processed for gene expression studies. Project Goal 3 - Coordinate evolution of photoperiodic regulation of flowering andcarbohybrate metabolism insunflower. We have completed growth chamber experiments where sunflower plants from a variety of cultivated and wild accessions that differ in their photoperiod responseare being raised under 1) long days, 2) short days, or 3) long days followed by transfer to short days after less than 2 weeks.Leaf and stem tissue was collected over a time course from pre- to post-flowering as is awaiting processing for carbohydrate analysis. Project goal 4 -Genetic of natural variation in critcal photoperiod in Mimulus. During the reporting period, we advanced germplasm resources for fine mapping and transcriptomic analyses. Specifically, we extended our four high x low elevation crosses to the F4 generation, and we now in the process of finishing collections of selfed seed for progeny testing and leaf tissue for genotyping from all panels. For the one panel that has moved forward more quickly than the other three, genotyping to identify which F4s arerecombinants within three critical photoperiod QTLs in that cross has been completed,and selfed progeny will be sown for progeny testing soon. NIL construction has gone more slowly than we would have liked, but it is progressing. We aim to have this completed in the next reporting period and then perform transcriptomics studies of the NILs in subsequent reporting periods. During this reportingperiod, we have also been finalizing experiments and analyses for our first major publication of our data on this topic. In this publication, we will report the QTL analysis of one cross; gene expression studies revealing that cis-regulatory differences affecting transcription of a tandem array of major flowering repressors is likely the primary cause underlying one of these QTLs; field studies showing that two of the QTLs for critical photoperiod have significant effects on flowering and/or fitness in high and low elevation native habitats; and population genomics studies thatrevealsignatures of selective sweeps coincident with the critical photoperiod QTL regions. With respect to the last finding, another key effort in the lab during this reporting period has been the successful adoption of a protocol for production of barcoded, shotgun sequencing libraries using microvolume amounts of the Illumina Nextera kit, essentially allowing us to produce 20x more libraries from this expensive commercial product. We used this method to generate the 40 individual genomiclibraries contributing to the manuscript in preparation, but we also plan to use this method to re-sequence 10-15 individuals from each 24 populations total sampled along two elevation gradients in the Cascades and Sierra Nevada mountain ranges. Doing so will alow us to identify genes for which allelic variation is distributed along environmental gradients, thus making them excellent candidates for adaptation to climate. We have made these same collections for five seasons now (including the one in the current period) to support future work examining how allele frequencies shift across time (including from the beginning to the end of the recent multi-yeardrought in the western US). Project Goal 5 - Flowering and fitness in native seasonal environments. Because NIL construction is still ongoing, we used the summer 2017 field season to grow the high and low elevation parent genotypes at high and low elevation sites in OR to do a pilot field study of gene expression in the flowering time gene regulatory network. We collected diurnal time courses ofleaf tissueharvested ~4 weeks after transplant into each site. This tissue will be processed for RNA extraction and transcriptomic studies in the next reporting period. Project Goal6 -Methods development for functional geneticsin sunflower and monkeyflower. We have now implemented and improved the protocol for transformation of the common monkeyflower,Mimulus guttatus, through tissue culture initially developed by the UC Davis Plant Transformation Facility. Notably, we are the first individual lab to have done so. We have used this method to express hairpin RNAs (hRNA) targeting several genes affecting nectar guide pigmentation patterning in flowers that have emerged after fine mapping studies to be strong candidates for natural variants affect this trait, and these knockdownstudies have confirmed that these loci are the causal genes. We have also generated transgenic lines carrying one or the other of two constructs carrying alternateallelic verisons of thepromoter region of one our these pigmentation candidate genes fused to the GUS gene. Once these mature to flowering, we will test the impact of the promoter allele onGUS expression intensity. We have also made slow but steady progress on implementing an existing but low efficiencyprotocol for sunflower transformation through tissue culture and prepared CRISPR/Cas9 constructs targeting the PDS gene for testing the efficacy of gene editing in planta and protoplast. As part of this work, we have adoptedan in vitro method for gRNA validation. In addition, we have sought (and now secured) gift funding from a plant biotechnology company to support a research associate in UC Berkeley's new Innovative Genomics Institute plant transformation facility to push these efforts forward full time starting from March 2018.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Blackman BK. Changing genes for changing seasons: natural variation in the plasticity of flowering time. Plant Physiology 173: 16-26 (2017).
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