Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Our target audience includes the plant science community, development agencies (United States Agency for International Development (USAID), USDA Foreign Agriculture Service (FAS) and others) and members of the chocolate industry. Changes/Problems:The COVID 19 pandemic has caused moderate slowdown of our project due to restrictions in lab access, slowdown in our outsourced services, necessity of visiting scientists to return home, and restrictions on meetings and travel. What opportunities for training and professional development has the project provided?The project supported the training and professional development of onegraduate student (Noah Winters, Penn State) and one postdoctoral scholar (Tuomas Hamala, Univ. of Minnesota) during the reporting period. These scientists have been integral in the planning and execution of all project activities. In addition, three technicians have participated in the project and gained experience in interdisciplinary and team research. We have involved one emeritus faculty member with expertise in statistics in the design and analysis of the transcriptome experiments. On average, we have eightjunior scientists in attendance at weekly or biweekly project meetings that include senior faculty from three universities, five departments, the USDA and sometimes two countries. How have the results been disseminated to communities of interest?Two scientists gave oral presentations at the Plant and Animal Genome meeting Jan. 2020. Two manuscripts have been published directly related to this work. Two additional manuscripts related to this work were published by project personnel. See publications section. A number of our base transient expression vectors and some containing cloned cacao genes have been made available to the research community via the Addgene company services. Websites: Project https://plantscience.psu.edu/research/labs/guiltinan/nsf-plant-genome-research-program Project Raw Data http://bigdata.bx.psu.edu/Cacao_NSF_data/ NCBI Bioproject https://www.ncbi.nlm.nih.gov/bioproject/558793 What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, under a no cost extension (already in place) we will complete all of our stated objectives and publish a series of major manuscripts. Comparative Genomics: We will complete the comparisons of genome structure variation in the new 37 genome assemblies with particular focus on regions identified to be associated with resistance and containing highly prioritized genes implicated in disease resistance. Population Genomics: Co-expressed modules of genes in T. cacao will be evaluated in relation to resistance/susceptibility and to divergence rates in those genes and flanking regions. Genomic analyses will be used to determine if macromutations that disrupt coexpression modules are associated with susceptibility. Genes differentially regulated in response to pathogen infection will be examined together with comparable data from root transcriptomes of 13 taxonomically diverse species of wild tropical trees. Conserved networks of co-expressed genes will be identified, along with defense responses unique to T. cacao. Functional Genomics: As stated above, a number of high priority candidate genes have been identified using our evolutionary genomics strategy and some of these have been cloned into expression vectors. We will continue transient expression/plant pathogen bioassays to assess the impact of over and/or under expression of these genes in plant defense. We will develop a simple graphical user interface for the image analysis system and make this code available to others in the field.
Impacts
What was accomplished under these goals?
Impact: Our work has impact to society by advancing our understanding of the mechanisms of plant growth and development and plant disease resistance. This is important to society because plants form the foundation of the food supply chain. Our work with cacao helps farmers in developing countries to earn a better living and supports a large US industry. Worldwide, approximately 5 to 6 million small-holder farmers grow 95% of the world'sproduction providing an important source of cash to otherwise primarily subsistence farmers. World cocoa export commerce is $5 to $6 billion/year and in the United States alone, the use of cocoa and cocoa butter in chocolate manufacturing, cosmetics, and other products drives an approximately $70 billionmarket providing over 60,000 jobs. In addition, US chocolate production also uses large amounts of sugar, nuts, and milk valued at approximately $3 billion/year in receipts to American farmers. Ecological Relevance: Cacao is grown in some of the world's most important biodiversity hotspots. With a long life-cycle, predominantly cultivated under shade trees, there are many environmental benefits of cacao cultivation such as watershed and soil conservation, provision of habitat for birds and other animals and provision of buffering habitat for protected wild rainforest. Our work also impacts society by training the next generation of plant scientists and educators. We work with undergraduate, graduate and postgraduate studies to advance their knowledge and capabilities to become independent scientists. Our past students work in industry, at universities and in government agencies providing the scientific workforce needed to develop sustainable agricultural systems. 1.(Guiltinan, Maximova, and Sheaffer) Major activities completed / experiments conducted; Transfer of Living Plant Collection: We have completed the importation (under strict USDA supervision and quarantine measures) of all of the genotypes used in this study. These plants are now growing at Penn State in a quarantine greenhouse and have been propagated through rooted cuttings. The plant identities were verified using single nucleotide polymorphism (SNP) markers to be sure no mislabeling happened during the transportation or propagation phases. After 2 years in quarantine, these plants can be made available to others for future studies. Large Transcriptome Experiment: We have completed the execution of a large experiment in which the plants understudy in this project were propagated and grown under greenhouse conditions and half infected with Phytophthora on developing leaves. RNA samples were obtained and are now being purified and prepared for transcriptome sequencing which will be performed in the next reporting period (mid-August 2020). This data will be used in a new round of candidate gene identification and prioritization and to identify modules of coregulated genes that may be involved in disease resistance. Completion of Genome Sequencing Objectives: In addition to the cacao accessions originally planned for this project, through National Science Foundation (NSF) approved research plan changes as a result of new sequencing capabilities, we were able to increase the number of genotypes to 37 and increase the quality of resulting assemblies considerably. A new hybrid assembly strategy was developed allowing us to combine data from different sequencing technologies, particularly valuable was the inclusion of data from 10x Genomics sequencing libraries. Data collected; Completed sequencing and assembly of 37 cacao genome (31 varying in resistance to Phytophthora and 6 additional genotypes of specific interests (VIP genotypes) 5 genomes were de novo assembled to very high-quality using hybrid datasets acquired using multiple sequencing technologies (Pacbio, Illumina, 10X libraries) (ave. contig L50 >200 kb, ave. scaffold L50 > 200 Mb). 32 genomes assembled to high confidence using only illumina data Transcriptome for Annotation of Genomes: In order to support the annotation of the new genomes, a transcriptome dataset from a set of pooled samples representing various tissue types and developmental stages were sequenced using standard Illumina Trueseq methods. Using this method we mapped >90% of conserved genes found in most plant species (BUSCOS) demonstratingvery high mapping completeness. All raw data and assemblies released on project website: https://plantscience.psu.edu/research/labs/guiltinan/nsf-plant- genome-research-program As a second site for data access to the public, a Bioproject was established at National Center for Biotechnology Information (NCBI) to provide access to all project data and metadata: Theobroma cacao (cacao): https://www.ncbi.nlm.nih.gov/bioproject/558793 Summary statistics and discussion of results; Population and evolutionary genomics analysis were performed on the new cacao datasets. Specific genomic intervals of interest were identified using a genome-wide association study (GWAS) approach to associate resistance to Phytophthora resistance phenotype with specific genic regions. A set of highly prioritized candidate genes have been identified based on expression characteristics and association with genomic regions exhibiting population-genetic signatures of adaptation. 2. (Guiltinan, Maximova, and Sheaffer) Please see above for major activities that also lead to this objective. We have discovered sequence variations in alleles of above mentioned genes that could make the basis for molecular markers once functions are fully characterized. We have contributed our datasets to the USDA Agricultural Research Service (ARS) which is a world leader in developing cacao genetic markers for breeding. 3. (Guiltinan, Maximova, and Sheaffer) Major activities completed / experiments conducted; We have developed a quarantine greenhouse section (within the goals of the project but with internal funding) that will support the growth and distribution of the germplasm we have sequenced. Thus, we have a paired set of genome assemblies and living cacao accessions that will be highly valuable for future studies. For example, currently one scientist is using these resources to study genetic variation for plant defense metabolites under separate funding. We have developed an automated phenotyping instrument and associated AI software to allow high throughput monitoring of plant pathogen infections. This technology and equipment will be made available widely to others and can be used in many different applications for automated phenotyping, etc. Data collected; (see above) Key outcomes or other accomplishments realized; We have developed techniques adapted for genome sequencing, gene editing, functional genomics in cacao and tissue culture and propagation methods and have shared these with other scientists around the world. 4. (Guiltinan, Maximova, and Sheaffer) One project scientist(Eric Wufula) has gained expertise in new genome assembly and annotation methods, namely 10X and hybrid 10X plus Illumina short read approaches. One of our former project participants is now working in industry and his contributions to the projects were foundational to the skills that made him competitive for the job. A graduate student in our project has obtained a USDA NIFA fellowship in which the project results and concepts contributed to the new line of research he will be conducting(comparative genomics of several related Theobroma species). Two postdocs are now employed externally, one at USDA and the second as a professor in Israel. One graduate student is now a postdoc. Approx. sixundergraduate students have gained experience in research by participation in various aspects of this project.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Maximova, S., and Guiltinan, M. (2018). The potential of somatic embryogenesis for commercial-scale propagation of elite cacao varieties. In achieving sustainable cultivation of cocoa, P. Umaharan, ed. (Burleigh Dodds Scientific Publishing). (https://shop.bdspublishing.com/store/bds/detail/product/3-190-9781838793548)
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Fister, A.S., Landherr, L., Perryman, M., Zhang, Y., Guiltinan, M.J., and Maximova, S.N. (2018). Glucocorticoid receptor-regulated TcLEC2 expression triggers somatic embryogenesis in Theobroma cacao leaf tissue. PLOS ONE 13, e0207666. Open Access Link
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Pokou, D.N., Fister, A.S., Winters, N., Tahi, M., Klotioloma, C., Sebastian, A., Marden, J.H., Maximova, S.N., and Guiltinan, M.J. (2019). Resistant and susceptible cacao genotypes exhibit defense gene polymorphism and unique early responses to Phytophthora megakarya inoculation. Plant Mol Biol (2019) 99: 499. https://www.ncbi.nlm.nih.gov/pubmed/30739243
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Gallego, A.M., Rojas, L.F., Rodriguez, H.A., Mora, C., Atehort�a, L., Urrea, A.I., Guiltinan, M.J., Maximova, S.N., Gaquerel, E., Zuluaga, M., et al. (2019). Metabolomic profile of cacao cell suspensions growing in blue light/dark conditions with potential in food biotechnology. Plant Cell, Tissue and Organ Culture (PCTOC). DOI 10.1007/s11240-019-01679-3. https://rdcu.be/bPOey
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Tuomas H�m�l�, Mark J Guiltinan, James H Marden, Siela N Maximova, Claude W dePamphilis, Peter Tiffin, Gene Expression Modularity Reveals Footprints of Polygenic Adaptation in Theobroma cacao, Molecular Biology and Evolution, msz206, https://doi.org/10.1093/molbev/msz206
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Progress 07/01/19 to 09/30/19
Outputs
Target Audience:The target audience is the plant science community, cacao genetics and breeding community, visiting scientists, and the US government. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Developed the Penn State Cacao and Chocolate Research Network, (CCRN) which brings together about 50 faculty and graduate students. CCRN has sponsored training workshops for all project participants in leadership, communication and grant writing. Through our NSF funded research project, we have offered two summer research fellowships to undergraduate minority students for the past two years and will host a third program in 2020. The students experienced lectures and hands-on research in the plant sciences at Penn State and at our field sites in Costa Rica and took classes and gained field experience at EARTH University in Costa Rica. How have the results been disseminated to communities of interest?Our results and activities are documented and distributed via peer review publications, scientific presentations and conferences, poster presentations at conferences and articles and interviews in the public press. We maintain active internet presence via a lab website, and active Facebook group. https://plantscience.psu.edu/research/labs/guiltinan Guiltinan - Maximova Labs Members, Alum, Collaborators and Friends https://www.facebook.com/groups/62535449847/ What do you plan to do during the next reporting period to accomplish the goals?During the coming year, we will focus much of our research effort on functional testing of the 10 candidate genes we have prioritized. We are also planning a large transcriptome experiment to explore the genetic variation between varieties and in response to pathogen infection. We have begun planning to write a new proposal as our NSF funding will expire in one year.
Impacts
What was accomplished under these goals?
This report covers only a few months but in this time we have made remarkable progress are goals 1, 3, and 4. During this timeframe, we have not addressed goal 2. The genome assemblies have greatly accelerated our ability to search for disease resistance genes (Goals 1, 3, and 4). Our candidate gene prioritization strategies have resulted in a list of ten genes of highest interest. A wide variety of over-expression and knockout transgenes are being constructed with these genes for functional analysis studies in arabidopsis and cacao(Goals 1 and 4). We have continued to develop CRISPR technology with cacao and to improve the cacao functional genomics pipelines(Goal 3). We have hosted several visiting scientists and are mentoring graduate students towards careers in the plant sciences(Goal4). We have supported the development of networks within Penn State and with US and international partners in industry, government and academia with a shared interest in the science and improvement of the cacao value chain and its impact on rural development and sustainable agriculture(Goal4).
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Andrew Fister, Mariela E. Leandro-Mu�oz, Dapeng Zhang, James H. Marden, Peter Tiffin, Claude dePamphilis, Siela N Maximova and Mark J. Guiltinan Wide ranging differences in tolerance to Phytophthora palmivora in four genetic groups of cacao. Tree Genetics and Genomes (Oct. 2019 in press).
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