Source: UTAH STATE UNIVERSITY submitted to
CYTOLOGICAL AND MOLECULAR CHARACTERIZATIONS OF REPRODUCTION IN SEXUAL AND APOMICTIC BOECHERA (BRASSICACEAE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1000552
Grant No.
(N/A)
Project No.
UTA01127
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 3, 2013
Project End Date
Jun 30, 2018
Grant Year
(N/A)
Project Director
Carman, J.
Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322
Performing Department
Plants, Soils & Climate
Non Technical Summary
Need for the project: Grains, principally rice and wheat, provide the majority of calories in the diets of people worldwide, ranging from 23% in the U.S. to 62% in Asia and North Africa. Yet yields for wheat and rice remain 15-30% below their biological potentials even when grown in ideal soils and climates using ideal irrigation and cultivation practices. Production of hybrid seed, which could close this gap, is not economically feasible for most major world crops including rice and wheat. In crops that are grown as hybrids, such as corn, high yields occur in the first season only. Hence, seed companies must produce hybrid seed every year, a process costing approximately $1 billion annually in the U.S. for corn alone. Additionally, the floral anatomy of wheat and rice prevents most seed companies from commercially producing hybrid seed of these crops. Such seed could be produced commercially if these plants were engineered to produce their seed clonally. Certain wild plants possess this capacity, including blackberries, dandelions, St John's wart, crab apples and Kentucky bluegrass. These plants are essentially high-yielding natural hybrids that pass hybrid vigor on to succeeding generations by asexual seed formation (apomixis). Apomixis does not occur in major world crops. One of our goals is to elucidate the molecular mechanisms of apomixis in wild plants. A second goal is to engineer crops with genes that may induce apomixis so that hybrids of these crops pass their hybrid vigor on through succeeding seed generations. This could make hybrid seed production feasible for essentially all crops. Methods and evaluation plan: Eight to 10 species of putatively sexual and putatively apomictic Boechera will be collected. Mode of reproduction will be evaluated cytologically by analysis of at least 50 appropriately staged ovules per species, to determine mode of embryo formation (milestone, Dec 2015), and by flow cytometry of at least 200 seeds per species, to determine mode of endosperm formation (milestone, Jun 2016). Genetic and epigenetic mechanisms of apomictic reproduction will be evaluated by sequencing small and large RNA pools from appropriately staged ovules of sexual and apomictic species and by identifying consistent differences among modes of reproduction in these RNA. These studies will provide information concerning the putative roles of genetics and epigenetics in the evolution and regulation of apomixis (milestone, Dec, 2017). Functional analyses of identified candidate genes will then be conducted by up and down regulating them transgenically in model species and in crop plants, the results of which may enable the engineering of crops that possess the agronomic impacts of apomixis as outlined above (milestone, Jun 2018).
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012499105060%
2022420104040%
Goals / Objectives
Goals and objectives: 1. Collect 8-10 putatively sexual diploid and 8-10 putatively apomictic diploid Boechera species and verify their reproductive mode (milestone, Dec 2015; deliverables, seed and living plant collections of verified sexual and apomictic Boechera) 2. Screen seeds of the apomictic Boechera for tolerance to exceptional maternal to paternal (MP) genome ratios in endosperm and for autonomous endosperm formation (milestone, Jun 2016; deliverables, verified modes of endosperm formation including possible departures from normal that could be of agronomic value) 3. Compare DNA and histone methylation levels in eggs and central cells of the 16-20 sexual and apomictic Boechera species (milestone, Dec 2016; deliverables, knowledge base concerning possible differences between apomicts and sexuals in DNA methylation of eggs and central cells) 4. Obtain deep sequence data for small and large RNAs during early ovule development and during early endosperm formation for sexual and apomictic Boechera (milestone, Dec, 2016; deliverables, large database identifying differences in gene expression during seed formation between sexual and apomictic Boechera) 5. Compare ovule and endosperm mRNA and sRNA of sexual and apomictic Boechera (milestone, Dec 2017; deliverables, evidence for the genetic and epigenetic causes of the differences identified in 4 above) 6. Verify gene expression in ovules and endosperm of 16-20 species and select candidate genes and gene networks (milestone, Dec 2017; deliverables, evidence-based rationale for proceeding to candidate gene analyses) 7. Examine candidate gene function (apomeiosis, parthenogenesis and autonomous or pseudogamous endosperm formation) in Boechera, Arabidopsis and sorghum (milestone, Jun 2018; deliverables, transgenic plants expressing apomixis or apomixis-like developmental processes)
Project Methods
Methods: (1) The identity of collected species will be accomplished using recently developed taxonomic keys and by microsatellite PCR. Mode of reproduction will be verified embryologically by DIC microscopy of cleared pistils and by single seed flow cytometry. Seed of all verified species will be maintained and made publically available through the Boechera seed resources network. (2) Sixty apomictic accessions, representing phylogenetic and ploidy diversity, will be analyzed for embryo and endosperm ploidy level by flow cytometry. Plants exhibiting autonomous endosperm will be grown, and flowers emasculated for verification. (3) Timing of endosperm formation in sexual Boechera and in apomictic Boechera capable of autonomous endosperm formation will be determined, and ovules will be analyzed cytologically to determine if differences in egg and central cell methylation (DNA and histone) exist between sexual and apomictic Boechera. Fluorescently labeled antibodies will be used. (4) Our previous studies suggest the regulation of apomeiosis, parthenogenesis and autonomous endosperm involve small RNA. Tissue for analyses will be collected at the pre-meiotic MMC to meiocyte stage (entire ovules), maturing embryo sac stage (entire ovules), and the mature embryo sac to early embryo and endosperm formation stage (embryo sacs micro-excised from ovules and divided into egg apparatus and central cell components). Illumina sequencing will be employed for large and small RNA analyses (collaborator). Ovules from a sexual control, a pseudogamous apomict, and a weakly to highly autonomous apomict will be used. sRNA ranging from 18-30 nt will be collected and unique RNA sequences that perfectly match the Boechera sequence will be subsequently analyzed. The eluted band library will be precipitated, re-suspended, and validated on an Agilent Bioanalyzer High Sensitivity Chip. Validated libraries will be pooled (48 indexes per pool) and clustered on an Illumina cBot. Amplified clusters will be sequenced on an Illumina GAIIx or HiSeq 2000 for 30 cycles, followed by a 6 cycle index read. Reads will be filtered for quality, trimmed to remove adapter, split on their index and Bowtie aligned to appropriate databases. (5) RNA sequence data will be analyzed, and comparative signatures of the major gene expression trends and changes in mRNA and sRNA between developmental stages and tissues will be obtained using appropriate bioinformatics tools. Such comparisons may allow key pathways to be identified and ultimately manipulated. (6) qPCR of mRNA and stem-looped primer qPCR of sRNA will be conducted using the resources of a collaborator, and the results of the qPCR analyses will be used to narrow in on a group of 15 to 25 mRNA or sRNA-based candidate genes. (7) Genes or pathways identified will be examined for cell type specific expression. This will utilize in situ and protein fusion marker analyses in transgenic plants as appropriate. Functional analyses of priority genes will be undertaken to determine their roles in apomixis. Specific promoters and Agrobacterium transformation will be used.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Public and private sector scientists interested in understanding differences in the molecular biology of sexual and asexual seed formation in plants. Changes/Problems:No major changes or problems. What opportunities for training and professional development has the project provided?Training of undergraduate and graduate students working in the lab involved the following topics: aseptic technique, fixation and clearing of floral samples for microscopy, differential interference contrast microscopy, aseptic technique, plant tissue culture experimentation, tissue culture media preparation, statistical procedures for analyzing large gene expression data sets, experimental design and statistical analysis procedures. How have the results been disseminated to communities of interest?A review seminar concerning the origins and molecular regulation of asexual reproduction through the germline in eukaryotes (apomixis) was prepared and presented at the Plant and Animal Genome Meetings and at four institutions in China. Recent project findings are awaiting patent evaluations and possible filings prior to public dissemination. What do you plan to do during the next reporting period to accomplish the goals?The scope of this project has largely been completed. During the remaining six months of the project, our efforts will focus on additional experimentation, preparation of manuscripts, and acquiring follow-on funding.

Impacts
What was accomplished under these goals? We completed cytoembryological assessments of reproductive mode for 87 Boechera taxa (species and hybrids). The original objective was to evaluate 20 taxa, but high variability for modes of reproduction (sexual and several types of apomixis) and mode penetrance required a more comprehensive survey. Most taxa exhibited high frequency apomixis with numbers of diplosporous and aposporous apomicts being nearly equal. Expression profiles of sexual and apomictic Boechera grown under different environmental conditions were analyzed, and experiments were conducted that altered environmental factors associated with mode of reproduction.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Carman, J. G., Invited Academic Presentation, "Apomixis in eukaryotes: an ancient fair-weather alternative to sex," Hunan Agricultural University, Changsha, China. (November 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Carman, J. G., Invited Academic Presentation, "Apomixis in eukaryotes: an ancient fair-weather alternative to sex," Hubei University. (November 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Carman, J. G., Invited Academic Presentation, "Apomixis in eukaryotes: an ancient fair-weather alternative to sex," Huazhong University of Science and Technology, Wuhan, China. (November 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Carman, J. G., Invited Academic Presentation, "Apomixis in eukaryotes: an ancient fair-weather alternative to sex," Hunan Academy of Forestry, Changsha, China. (November 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Carman, J. G., Invited Presentation, "Progress toward producing apomictic crops," Pioneer Hi-Bred Seed Company, Johnston, Iowa. (January 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Carman, J. G., International Plant & Animal Genome XXIV Conference, "Apomixis in eukaryotes: an ancient fair-weather alternative to sex," San Diego. (January 17, 2017)


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target Audience Plant scientists interesting in understanding the biochemical and molecular biology of the apomixis/sex switch in facultatively-apomictic angiosperms and seed companies interested in using apomixis for clonal seed-propagation of hybrid vigor. Changes/Problems:Changes/Problems No major changes or problems What opportunities for training and professional development has the project provided?Opportunities Graduate and undergraduate students learned biochemical pathways associated with apomixis/sex switching, design of experiment principles, and the importance of control groups and statistical analyses How have the results been disseminated to communities of interest?Dissemination Contents of a manuscript are currently being evaluated for patent protection prior to manuscript submittal. Three undergraduate students presented posters highlighting their findings at USU undergraduate and graduate student research symposia. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work We will continue pharmacological experiments aimed at dissecting and elucidating biochemical and molecular components of the apomixis/sex switch in angiosperms.

Impacts
What was accomplished under these goals? Accomplishments Twenty-two inter-related Boechera taxa were studied embryologically. Among these, we observed sexual reproduction and three types of apomictic reproduction (Antennaria and Taraxacum-type diplospory and Hieracium-type apospory. Eight taxa consistently produced genetically-reduced embryo sacs from surviving megaspores of meiotic tetrads, which indicates that they may be obligately sexual. The remaining taxa were apomictic with various ratios of apomictic to sexual embryo sac formation occurring. Many taxa exhibited high-frequency Taraxacum-type diplospory and/or Hieracium-type apospory. Antennaria-type diplospory was only infrequently observed and occurred only in B. retrofracta x stricta. Pseudogamy (central cells of apomictic embryo sacs requiring fertilization for seeds to form) was confirmed by single seed flow cytometry.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Carman, J. G., Seminars on Advances in the Genetic and Molecular Characterization of Apomixis V, "Effects of stress on reproductive mode in Boechera (Brassicaceae)," Argentine National Scientific and Technical Research Council (CONICET), Center for Natural Resources in the Semi-Arid Zone (CERZOS), Bahia Blanca, Argentina. (November 14, 2016 - November 16, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Carman, J. G. (Presenter & Author), Mateo de Arias, M. (Author Only), Nelson, S. M. (Author Only), Zhao, X. (Author Only), L. G. (Author Only), M. S. (Author Only), Sherwood, D. A. (Author Only), International Plant & Animal Genome XXIII Conference, "Hot on the trail of the sex-apomixis switch in Boechera (Brassicaceae)," San Diego, CA. (January 2015 - Present)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Carman, J. G., Seminars on Advances in the Genetic and Molecular Characterization of Apomixis V, "Apomixis in eukaryotes: an ancient fair-weather alternative to sex," Argentine National Scientific and Technical Research Council (CONICET), Center for Natural Resources in the Semi-Arid Zone (CERZOS), Bahia Blanca, Argentina. (November 14, 2016 - November 16, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Price, B. J. (Presenter & Author), Gao, L. (Author Only), Sherwood, D. A. (Author Only), Watts, L. (Author Only), Wright, D. (Author Only), Cox, B. (Author Only), Carman, J. G., Summer Undergraduate Biotechnology Internship Program, "Stress signals induce meiosis in apomictic Boechera (Brassicaceae)," Utah State University, Logan, UT. (August 11, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Anbesse, A. (Presenter & Author), Bogoev, Y. (Presenter & Author), Bryan, N. (Presenter & Author), Carman, J. G. (Author Only), Summer Biotechnology Academy (High School), "Reproductive development in apomictic plants following various treatments of stress," Utah State University, Logan, UT. (July 15, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Carman, J. G. (Guest Speaker), Mateo de Arias, M. (Author Only), Shilling, M. (Author Only), Sherwood, D. A. (Author Only), Dwivedi, K. K. (Author Only), Srivastava, M. K. (Author Only), Lawit, S. J., Components of Apomixis Workshop, "A stress-induced polyphenic switch from apomeiosis to meiosis occurs in Boechera (Brassicaceae) that is cytologically and molecularly comparable to those of other kingdoms.," International Plant & Animal Genome XXIV Conference, San Diego, CA. (January 9, 2016 - January 13, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Albertini, E. (Guest Speaker), Carman, J. G. (Author Only), Shrivastava, M. K. (Author Only), Components of Apomixis Workshop, "DNA Methylation events specific of apomictic lineages in the Boechera genus," International Plant & Animal Genome XXIV Conference, San Diego, CA. (January 9, 2016 - January 13, 2016)


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target Audience i) Plant taxonomists and geneticists interested in modes of asexual (apomictic) reproduction expressed in Boechera (Brassicaceae), ii) academic and seed industry geneticists interested in the genetic and epigenetic regulation of apomixis and using apomixis to fix hybrid vigor in crops Changes/Problems:Changes/Problems No changes or serious problems. What opportunities for training and professional development has the project provided?Opportunities A phenomenal opportunity emerged in 2014 with two discoveries: 1) the apomeiosis pathway in Boechera can be switched efficiently to meiosis through drought and heat stress, and 2) this apomixis/sex switching is achievable in vitro using pharmacological chemicals. In 2015, we verified by RNASeq that the stress-induced morphological switch from apomeiosis to meiosis is accompanied by global shifts in gene expression, which cause the transcriptomes of ovaries from apomictic plants to closely resemble those of sexual plants in terms of gene ontology categories. This discovery implies that apomixis in Boechera, and probably in most other apomictic angiosperms, is an epigenetically-determined, potentially conserved, polyphenism of sex - not a genetically-determined, mutation-based trait. This discovery could have profound implications in terms of eventually harnessing apomixis, through transgenic modifications, for crop improvement, e.g., perpetuating hybrid vigor by asexual seed formation in currently-hybrid crops and in crops not currently produced as hybrids because of prohibitive costs associated with producing hybrid seed. Our research collaborations, involving Duke University, University of Perugia (Italy), Indian Grassland and Fodder Research Institute (India), DuPont Pioneer Hi-Bred, and the USU Biology Department, have represented important opportunities through which useful information has been exchanged and through which training opportunities have occurred. How have the results been disseminated to communities of interest?Dissemination Dissemination of research findings this year consisted of two peer-reviewed journal article publications, four presentations at international plant-biology conferences, and collaborative exchanges of data with scientists from Duke University, University of Perugia (Italy), the Indian Grassland and Fodder Research Institute (Jhansi, India), the USU Biology Department, and the international seed company DuPont Pioneer Hi-Bred, Inc. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work The first two years of this project generated lots of data in all seven objective areas, most of which were analyzed in 2015. Except for publication, Objective 1 is largely complete. Objective 2 includes screening seeds of many different apomictic Boechera species with the hope of identifying apomicts that produce endosperm autonomously (without fertilization of the central cell), i.e., having a 2C:4C embryo to endosperm genome ratio. So far, the apomictic Boechera we have tested exhibit a 2C:5C or 2C:6C ratio, which means endosperm only forms when the embryo sac central cell is fertilized. For crop improvement applications, autonomous endosperm formation is preferred. We will continue screening additional Boechera in an effort to identify autonomous apomicts. The DNA methylation and RNASeq studies of whole pistils conducted in 2014 and analyzed in 2015 have provided justification for conducting these types of analyses using single cells. A research proposal is being prepared to provide funding for these activities. Our major goal for 2016 is to conduct detailed pharmacological experiments to dissect and elucidate signal transduction pathways responsible for maintaining apomictic reproduction, epigenetically shifting apomictic to sexual reproduction, maintaining sexual reproduction, and epigenetically shifting sexual to apomictic reproduction.

Impacts
What was accomplished under these goals? Accomplishments Accomplishments by major goal: Objective 1 - Boechera germplasm collection and verification. Goal: collect seed and verify reproductive mode for 8-10 sexual and 8-10 apomictic diploid Boechera species. This was largely completed in 2014. In 2015, we continued to characterize mode of reproduction by cytology and flow cytometry of single seeds. These activities provided learning opportunities for three undergraduate and three graduate students, and the results are being prepared for publication. Objective 2 - mode of endosperm formation studies. In 2015, we continued to verify mode of endosperm formation in our apomictic Boechera by obtaining embryo-endosperm genome ratios of individual seeds. These activities provided learning opportunities for three undergraduate and three graduate students, and the results are being prepared for publication. Objective 3 - DNA and histone methylation levels. This objective was indirectly worked on in 2015 (see objective 5). Objective 4 - Deep sequencing of mRNA and sRNA during early ovule and endosperm development. Deep sequencing of mRNA from pistils of well-watered and droughted plants of one sexual, one facultatively-apomictic (sex and apomixis occurring simultaneously in different ovules of the same plant), and two mostly apomictic plants was completed in 2014 by an industry collaborator, and these data were analyzed in 2015. An overview of the results is presented in the objective 5 section. Objective 5 - compare ovule and endosperm transcriptomes among sexual and apomictic Boechera. In many eukaryotic apomicts, stress activates an epigenetic switch from apomixis (unreduced gamete formation and parthenogenesis) to sex (meiotic gamete formation and syngamy). For this to occur, a third polyphenic gender, an apomictic female, is often observed. She is well adapted to favorable conditions, is adept at producing clonal progeny, and, though genetically identical, often differs phenologically and morphologically from sexual females. Stress-activated epigenome reprogramming occurs in her progeny during their development such that sexually-functional males and females form. The genetically-identical siblings often mate to produce fertilized spores or eggs, which tolerate the stresses that induced their formation, i.e., short photoperiods, cold, desiccation, etc. When favorable conditions return, apomictic females form from the fertilized spores or eggs. This cyclical apomixis pattern occurs in animals, fungi, protists, diatoms, and brown and green algae. In apomictic flowering plants, polyphenic genders are not recognized. Instead, apomixis is generally facultative, with seeds often forming sexually and apomictically on the same plant. In 2015, we documented for Boechera (Brassicaceae) stress-induced switching from mostly apomeiotic to mostly meiotic ovule development, and we showed, by RNASeq studies, that this switching includes global epigenetics-based changes in gene expression that produce gene ontology patterns typical of those observed during reproduction in obligate sexual plants. These findings unify angiosperms with other eukaryotes in terms of i) tendencies for stress-induced polyphenic reversions from apomixis to sex and ii) physiological adaptations in apomicts that maximize fecundity thus facilitating geographic parthenogenesis. A primitive origins hypothesis for sex-apomixis polyphenisms, with functionality more-or-less conserved among eukaryotes, is supported. Objective 6 - verification of expression profiling data. In 2014, we used qPCR to verify differential expression of 21 genes from pistils of sexual versus pistils of apomict Boechera as determined previously in our lab by microarray analyses. In 2015, we further compared the qPCR results with RNASeq results. In all cases, results were consistent in direction of differential gene expression. Objective 7 - functional analyses. In 2015, we further developed and tested an in vitro model system for testing effects of environmental signals or transgenic alterations on shifts in reproductive mode of sexual and apomictic Boechera. This system will be used in future functional analyses of candidate genes that putatively control the sex apomixis switch in Boechera.

Publications

  • Type: Other Status: Published Year Published: 2015 Citation: Refereed Journal Articles Windham, M. D., Beck, J. B., Lei, F.-W., Allphin, L., Carman, J. G., Sherwood, D. A., Rushworth, C. A., Sigel, E., Alexander, P. J., Bailey, C. D., Al-Shehbaz, I. A. (2015). Searching for Diamonds in the Apomictic Rough: A Case Study Involving Boechera lignifera (Brassicaceae). Systematic Botany, 40(4), 1-15.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Carman, J. G. (Presenter & Author), Mateo de Arias, M. (Author Only), Nelson, S. M. (Author Only), Zhao, X. (Author Only), L. G. (Author Only), M. S. (Author Only), Sherwood, D. A. (Author Only), International Plant & Animal Genome XXIII Conference, "Hot on the trail of the sex-apomixis switch in Boechera (Brassicaceae)," San Diego, CA. (January 2015 - Present)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Srivastava, M. K. (Presenter & Author), Mateo de Arias, M. (Author Only), Sherwood, D. A. (Author Only), Carman, J. G. (Author Only), XXIII International Grassland Congress, "Status and role of antioxidant enzymes in sexual and apomictic species of Boechera," New Delhi, India. (November 20, 2015 - November 24, 2015)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Albertini, E. (Presenter & Author), Bocchini, M. (Author Only), Srivastava, M. (Author Only), Carman, J. G. (Author Only), 11th International Congress of Plant Molecular Biology, "DNA methylation changes conserved across apomictic species of the genus Boechera," Bourbon Cataratas Convention & Spa Resort, Iguazu Falls, Brazil. (October 25, 2015 - October 30, 2015)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Horandl, E., Hojsgaard, D., Klatt, S., Carman, J. G., Botanikertagung 2015, From Molecules to the Field, "The significance of apomixis for evolution and biodiversity of angiosperms," Ludwig-Maximilians-Universit�t & Technische Universit�t M�nchen, Munich, Germany. (August 30, 2015 - September 3, 2015)


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target Audience Primary focus groups for research conducted this year include academic scientists interested in evolution and genetic regulation of apomixis and commercial biotechnologists interested in using apomixis for fixing hybrid vigor in crops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Opportunities A phenomenal opportunity emerged this year with two discoveries: 1) the apomeiosis pathway in Boechera can be switched efficiently to meiosis through drought and heat stress, and 2) this apomixis/sex switching is achievable in vitro using pharmacological chemicals that (a) mimic heat or drought stress and (b) interrupt or trigger specific metabolic pathways. We now have an experimental system that should be very useful to us in molecularly characterizing apomixis. Our research collaborations, involving Duke University, University of Perugia, Indian Grassland and Fodder Research Institute (India), Pioneer Hi-Bred, and the USU Biology Department, have also represented important opportunities through which useful information has been exchanged and through which training opportunities have occurred. Wor45k on Objective 1 provided research and training opportunities for three PhD students (Mayelyn Mateo de Arias, David Sherwood, Lei Gao), a visiting PhD scientist from India (Manoj Srivastava), a visit lecturer from China (Xinghua Zhao), and an undergraduate USU Presidential Scholar (Susan Nelson). Mayelyn is assisting in preparing the manuscript, which will detail modes-of-reproduction we found in Boechera and correlate them with the Boechera phylogenetic tree. The completion of Objective 2 provided training opportunities for Mayelyn, David, Lei and Manoj, and Mayelyn is currently preparing a manuscript as part of her dissertation that includes these data. Work on Objective 3 provided training opportunities for Manoj, Mayelyn and David. Work on Objective 4 is an adjunct to Mayelyn's PhD research, and it is also providing training opportunities for large data set analyses for those working in my lab and, through collaboration, in Paul Wolf's lab, USU Biology Department. Martin Schilling, one of Paul's graduate students, is taking an active role in the data analysis phase. Work on Objective 6 provided training opportunities for Manoj and Mayelyn. Objective 7 is part of Mayelyn's dissertation and is currently being prepared for publication. Additional experiments conducted by my PhD student David Sherwood showed that apomeiosis in pistils of apomictic Boechera cultured in vitro can be switched to meiosis upon exposure to pharmacological chemicals specific to certain metabolic pathways. How have the results been disseminated to communities of interest? Dissemination Dissemination of research findings this year consisted of two peer-reviewed publications and collaborative exchanges of data with scientists from Duke University, University of Perugia (Italy), the Indian Grassland and Fodder Research Institute (Jhansi, India), the USU Biology Department, and the seed company DuPont Pioneer Hi-Bred, Inc. What do you plan to do during the next reporting period to accomplish the goals? Plan of Work The first full year of this project generated lots of data in all seven objective areas, which in 2015 will be more fully analyzed and disseminated. Except for publication, Objective 1 is largely complete. Objective 2 includes screening seeds of many different apomictic Boechera species with the hope of identifying lines that produce endosperm autonomously, i.e., have a 2C:4C embryo:endosperm genome ratio. So far, the apomictic Boechera we have tested exhibit a 2C:5C or 6C ratio, which means endosperm only forms when the embryo sac central cell is fertilized. For applications in crop improvement, autonomous endosperm formation is preferred. We will continue screening additional Boechera in an effort to identify autonomous apomicts. The DNA methylation and RNASeq studies of whole pistils conducted in 2014 as part of Objectives 3-5 should provide justification for these types of analyses but conducted on single cells. Additional funding, either Federal or private, will be required to conduct these studies and will be requested. Meanwhile, we will continue our physiological and biochemical manipulations of the apomixis pathway as guided by our previous expression profiling results.

Impacts
What was accomplished under these goals? Accomplishments Accomplishments by major goal: Objective 1 - Boechera germplasm collection and verification. The goal here was to collect seed and verify reproductive mode for 8-10 sexual and 8-10 apomictic diploid Boechera species. With the collections made this past summer, we have now completed this objective. Our collection includes seeds of ploidy-verified and reproduction-verified sexual diploids of B. crandallii, B. exilis, B. formosa, B. imnahaensis, B. lincolnensis, B. retrofracta, B. stricta, and B. yellowstonensis and seeds of apomictic diploids and/or triploids of B. duchesnensis, B. formosa hybrid, B. gunnisoniana hybrid, B. lignifera, B. lincolnensis hybrid, B. microphylla, B. microphylla hybrid, B. oxylobula hybrid, B. pallidifolia hybrid, B. pendulina hybrid, B. retrofracta x exilis, B. retrofracta x stricta, and B. thompsonii. Additionally, we verified reproductive mode in chemically-fixed flower samples obtained from Michael Windham (additional accessions), Duke University. Species involved included sexual B. dispar, B. exilis, B. exilis x retrofracta, B. formosa, B. inexpectata, B. kelseyana B. lemmonii, B. oxylobula, B. pendulina, B. schistacea, and B. sparsiflora and apomictic B. crandallii, B. gracilipes, B. lavigata, B. pulchra, B. retrofracta and B. thompsonii. We discovered that apospory, a form of apomixis not previously documented in Boechera, is common in the genus, occurring frequently in B. crandallii, B. gracilipes, B. lavigata, B. lignifera, B. lincolnensis hybrids, B. microphylla, B. pulchra, B. retrofracta hybrid and B. thompsonii hybrids. Apospory may be more common in Boechera than the previously-documented apomictic type, diplospory. Objective 2 - mode of endosperm formation studies. The embryo:endosperm genome ratio in seeds of most sexual plants is 2:3, but in apomictic plants it is 2:4, 5 or 6. Hence, it is possible to use a flow cytometer to determine mode of reproduction simply by obtaining DNA content values of individual seeds for the embryo and endosperm. The technique becomes tricky with non-endospermic species, e.g., Arabidopsis and Boechera, which consume most of their endosperm early in seed fill. Nevertheless, by using mortars and pestles with brief and gentle grinding, we were successful in obtaining endosperm values from individual seeds for most of our Boechera species. Importantly, we observed that B. microphylla, previously thought to reproduce sexually because of reduced pollen, actually reproduces apomictically (2C:5C embryo:endosperm ratio). Being able to document mode of reproduction based on embryo and endosperm analyses of single seeds (Objective 2) was essentially achieved in 2014. This technique will be used routinely for completing additional objectives. Objective 3 - DNA and histone methylation levels. In collaboration with Emidio Albertini, University of Perugia, Italy, the methylation status of young pistils excised from sexual B. imnahaensis, B. retrofracta, B. stricta and B. yellowstonensis and apomictic B. lignifera, B. retrofracta x exilis, B. retrofracta x stricta and B. microphylla was assessed. Twelve primer combinations were used to assay cytosine methylation at 5'-CCGG-3' sequences in the eight species. A total of 1203 reproducible bands were amplified. The extent of DNA methylation ranged from 70.55% in B. retrofracta to 83.54% in B. yellowstonensis. We found 100 and 114 polymorphisms between apomictic and sexual species for the Hpa and Msp restriction enzymes, respectively. This may reflect epigenetic differences in gene regulation between modes of reproduction. Ten polymorphisms were shared between apomictic and sexual genotypes. These are now being sequenced and will be compared to the publically available B. stricta JGI genome sequence. Genes determined to be differentially regulated epigenetically, between apomicts and sexuals, will be compared with differentially expressed genes previously identified by our RNASeq and microarray studies. Our results were obtained using entire pistils, which include many tissue types. Finding differences between sexuals and apomicts at this heterogeneous tissue level may provide a strong rationale to compare DNA methylation levels of eggs and central cells (precursors of endosperm) of apomicts with those obtained from sexuals. Objective 4 - Deep sequencing of mRNA and sRNA during early ovule and endosperm development. We conducted experiments to determine effects of plant stress (drought and drought plus heat) on frequency of apomixis (apomeiosis and parthenogenesis) in several Boechera species. As part of the experiment, RNA was collected from immature pistils of stressed and non-stressed apomictic and sexual plants. The mRNA from these samples was sequenced (Illumina cBot) by a collaborator and aligned to the publically-available B. stricta genomic sequence (Department of Energy, Joint Genome Institute, 278 Vol. 1.2). Objective 5 - compare ovule and endosperm transcriptomes among sexual and apomictic Boechera. In our RNASeq experiments, over 4000 differentially expressed transcripts were identified between immature pistils from sexuals and apomicts. Once fully analyzed, we expect this experiment to provide justification for more detailed studies in which gene regulation, and methylation status, will be studied at the germline and endosperm levels in sexuals and apomicts. Objective 6 - verification of expression profiling data. We used qPCR to verify differential expression of 21 genes from pistils of sexual versus pistils of apomict Boechera as determined previously in our lab by microarray analyses. In all cases, qPCR and microarray analyses were consistent in direction of differential gene expression. Genes tested were AT2G48020, AT3G13580, AT3G52940, AT4G02730, AT2G40140, At2g28190, At4g08390, At1g77490, At2g25080, At4g31870, At3g11630, At5g06290, At3g52960, At3g26060, At4g35000, At1g20620, At3g10920, At2g28190, At3g18780, At2g44120 and At3g13580. Objective 7 - functional analyses. Our drought and heat-stress experiments were conducted to evaluate putative shifts in reproduction (from apomictic to sexual) and to evaluate expression of candidate genes. According to our hypotheses, stress can trigger shifts from asexual apomeiosis, in which genetically-unreduced dyads form, to sexual meiosis, in which genetically-reduced tetrads form. Under favorable growth conditions, dyads (mostly diplosporous) in apomictic Boechera were observed 5.3 to 100-fold more frequently than sexual tetrads (depending on species facultativeness). In sexual species, tetrads were observed 20-fold more frequently than dyads. The latter occurs because the dyad stage of meiosis in sexuals is very brief, while the tetrad stage is lengthy. In obligate diplosporous species, tetrads do not form, only dyads. When apomictic Boechera were drought stressed, dyads occurred only 1.6 to 3.8-fold more frequently than sexual tetrads, i.e., sexual meiosis became much more frequent. Furthermore, when apomictic plants were both drought and heat stressed, sexual tetrads formed 2 to 3-fold more frequently than diplosporous dyads. This large-scale switching from apomeiosis to meiosis was correlated with an increase in the expression of stress response genes. However, under stress, ovule development generally aborted (ovules failed to produce seeds). Based on single-seed flow cytometry, all seeds developing to a viable stage developed apomictically. This suggests that the sexual pathway, though triggerable by stress, is not viable in the apomictic Boechera studied, i.e., wide hybrids and triploids that generally are sexually sterile. Thus, mutations, chromosome aberrations or genetic recombinations that occurred after the evolution of apomixis in Boechera and that enhance apomixis frequency are likely to be strongly adaptive.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Dwivedi, K. K., Roche, D., Clemente, T. E., Ge, Z., Carman, J. G. (2014). The OCL3 promoter from Sorghum bicolor directs gene expression to abscission. Annals of Botany, 114, 489-498. http://aob.oxfordjournals.org/
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Hojsgaard, D., Klatt, S., Baier, R., Carman, J. G., Horandl, E. (2014). Taxonomy and biogeography of apomixis in angiosperms and associated biodiversity characteristics. Critical Reviews in Plant Sciences, 33(5), 414-427. http://www.tandfonline.com/toc/bpts20/current#.VJCQqD90zRY


Progress 09/03/13 to 09/30/13

Outputs
Target Audience: Plant biology and seed industry communities Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? David Sherwood had the opportunity to spend 11 days this past summer at DuPont Pioneer Hi-Bred Company in their molecular biology facility designing and building gene constructs for a collaborative research project we have with Pioneer that is directly related to this project. Two visiting molecular biologist senior scientists from India, Dr. Krishna Dwivedi (three months) and Dr. Manoj Srivastava (12 months, returns to India 11, 2014), joined my lab this year to conduct collaborative research. This has provided learning opportunities for myself and my three graduate students as well as for the visiting scientists. I attended the European Frontiers of Plant Reproduction Research 2013 conference in Oslo, Norway as an invited speaker. This conference provided learning opportunities and an additional opportunity to establish a new research collaboration. How have the results been disseminated to communities of interest? This new project that has not yet generated publishable information. What do you plan to do during the next reporting period to accomplish the goals? In 2014, we plan to initiate the following experiments: (i) transformation of Arabidopsis and Boechera with constructs produced during 2013-14 (Objective 6-7), (ii) validate using qPCR gene expression patterns obtained by microarray and determine effects of stress on differential gene expression in ovules between sexual and apomict Boechera (Objectives 3-6), (iii) explore REDOX signaling during meiosis in sexual Arabidopsis and in sexual and apomictic Boechera (Objective 7), (iv) conduct methylation sensitive amplified fragment polymorphism experiments to evaluate the possible levels of epigenetic differentiation at the genome level in ovules between sexual and apomictic Boechera (Objective 3), and (v) continue embryological analyses of sexual and apomictic Boechera species as part of a taxonomic treatment of Boechera (Objective 1).

Impacts
What was accomplished under these goals? Accomplishments by major goal: Objective 1 - Boechera germplasm collection and verification. Seven of 8-10 sexual and six of 8-10 apomictic diploid Boechera species proposed for collection and verification were collected and verified embryologically (see Physical Collection). Objective 2 - mode of endosperm formation studies. The embryo:endosperm genome ratio in sexual plants is 2:3. In apomictic plants, this ratio is usually 2:5 or 2:6 if fertilization is required for endosperm formation (pseudogamous endosperm). It is 2:4 if fertilization is not required for endosperm formation (autonomous endosperm). The latter is most attractive for agriculture. Flow cytometry is used to determine genome ratios from single seeds. We modified techniques to obtain genome ratios from single seeds using our flow cytometer. Objective 3 - DNA and histone methylation levels. An external collaboration was established to determine extent of DNA and histone methylation during germline development in sexual and apomictic Boechera. Plants for this collaboration have been sown. Objectives 4-5 - no activity this year. Objective 6 - verification of expression profiling data. Eight genes putatively involved in differentiating sexual from apomictic development during seed formation have been identified based on previous expression profiling work in our lab. PCR primers have been selected based on Arabidopsis sequence information, and these are currently being tested using Boechera gDNA prior to use in qPCR verification of expression profiling data. Objective 7 - functional analyses. In collaboration with an external partner, genetic constructs for 10 of about 22 candidate genes have been engineered with appropriate promoters into Agrobacterium tumefaciens, and these will be used to up or down-regulate candidate genes in sexual Arabidopsis and apomictic and sexual Boechera. (1) Major activities completed: Major tasks largely completed during the first six months of the project include: germplasm collection with associated taxonomic and embryological verification, development of methods for single seed flow cytometry, and building of genetic constructs for genetic engineering. Other activities that the PI contributed to as a co-investigator, which are closely associated with the present project, include: (i) completion of an updated inventory of apomixis in angiosperms, (ii) preparation of a database listing apomictic angiosperms and definitive literature for each (online), (iii) preparation of a publication related to the database (submitted), and (iv) preparation of a manuscript describing the function of an ovule-specific promoter important to seed fill in cereals (in final revision prior to submittal). (2) Specific objectives met: This is a new project. Only the first objective, as discussed above, is close to being completed. (3) Significant results achieved: The ability to determine in-house embryo:endosperm genomic ratios using our flow cytometer is a technical development that will save us time and money in obtaining our research objectives. We had previously planned to send a student to an external lab to conduct these analyses. (4) Key impacts or other accomplishments realized: This is a new project and has not yet generated key impacts. However, funding of this project is allowing us to meet research expectations with collaborators at four external public and private institutions and publication expectations with collaborators at four additional public institutions.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Carman, J. G. (Keynote Address), European Frontiers of Plant Reproduction Research 2013, "Apomictic life cycles: ancient fair-weather alternatives to sexual life cycles," European Cooperation in the field of Scientific and Technical Research (COST), Oslo, Norway. (October 2, 2013 - October 4, 2013)