Progress 08/15/24 to 08/14/25
Outputs
Target Audience:To reach my target audiences, I am communicating research discoveries and methods by publishing my results and presenting at scientific conferences as well as communicating results to conservation managers who can distribute results to farmers in a way that helps them grow higher-value cacao. As a teaching assistant I am educating undergraduate students in the U.S. about where their food, especially chocolate, comes from and the value of farmers and the food supply chain. Additionally, I am communicating my results to the people who manage the cacao research station where the research was performed. They are working to create practical agroforestry techniques to improve cacao cultivation. BFREE is interested in Criollo cacao because it is a valuable andlocally importantcacao found in Belize that farmers could grow to increase their income, while providing a highly sought after cacao for the craft chocolate industry. Changes/Problems:Unexpected outcome: Parentage analysis has been complicated due to the population having potentially clonal population structure or high levels of inbreeding. These relationships are confounding kinship analyses, and steps are being taken to ensure the data is analyzed correctly given this unexpected finding. Once the population structure is determined, further parentage analyses will be performed. If the parents are clonal or highly related, it will be difficult to confidently identify pollinating trees, thus making the parentage analysis impossible. Although this would be unfortunate when viewed from the original objectives of the project, it would be an interesting discovery to report and would provide crucial information for breeding efforts that involve Criollo cacao and provide useful information on the evolutionary history of and past human utilization of cacao. Although these outcomes have been unexpected, they have not substantially impacted the current rate of expenditure to date. Although some seedlings have been sequenced and will be used for population genetic analysis, further sequencing of seedlings has been paused until we are confident that the sequencing will provide useful information. Travel to collect more samples has been delayed until the genetic structure of the population is finalized to ensure that funds are spent wisely to further our understanding of the subject. Any substantial changes will be communicated to USDA-NIFA. What opportunities for training and professional development has the project provided?I wrote and was awarded an internal proposal to hire and mentor a University of Tennessee Institute of Agriculture undergraduate student to assist with insect DNA extraction and sequencing. The undergraduate student is studying horticulture in the Plant Sciences Department and is considering pursuing a graduate degree in plant science. Her work in the lab is providing her an opportunity to discover if this area of research sparks her interest. She is also gaining experience that will be crucial for successfully obtaining future work opportunities. Additionally, I am gaining additional mentoring experience which will prepare me for future roles, all while receiving help to complete the project in a timely manner. I participated as a TA for an undergraduate class on chocolate, EPP123, Chocolate: Bean to Bar. This class educates students on where their food comes fromand the importance of agricultural supply chains. I also participated in teaching a lecture for an RNA-seq short course to undergraduate and graduate students. Additionally, I helped mentor a fellow University of Tennessee graduate student with research, writing, and preparing to defend his thesis. The topic of his research was on the fungal communities found in the same Criollo cacao trees that are being studied as part of my USDA-NIFA pre-doctoral project. He successfully defended his thesis and has acquired full-time employment in agriculture as a growing specialist in Monroe, Georgia. His research and connections have helped to disseminate information about both his and my research to farmers currently growing cacao in Belize. I participated as a peer reviewer for three journals. These included Ecology and Evolution, Open Life Sciences, and the Brazilian Archives of Biology and Technology. Two of the articles focused on cacao, and one of the articles was on plant genetics more broadly. I assisted with planning and coordinating the Tennessee Plant Research Center's monthly colloquia held at the University of Tennessee. This includes coordinating invited researchers to present their research and collaborate with University of Tennessee research groups. How have the results been disseminated to communities of interest?Current progress and results weredisseminated atconferences. I attended two conferences this summer to present my research and collaborate with other researchers. I first attended Evolution in Athens, GA. I then attended Botany in Palm Springs, CA. Discussions resulting from my presentations have provided valuable direction to my project from outside perspectives. Preliminary results have been communicated to the directors of the BFREE field station on several occasions. They are motivated to use information generated to implement any improvements in their efforts to study and promote cacao agroforestry. I am also communicating with the Ya'axché Conservation Trust. They can provide information about cacao research to local farmers. To increase public awareness about the research and educate the public on the issues surrounding cacao production, an article about the project was published in the University of Tennessee's magazine Life, Land, & Science. What do you plan to do during the next reporting period to accomplish the goals?Work to complete sequencing and data analysis for all cacao trees, seedlings, and insects is underway. Once analyses are complete, publications will be drafted and submitted to appropriate journals. Mentoring of U.S. undergraduate students in research and scientific presentation will continue, and further updates to BFREE directors and other invested groups will occur. Committee meetings will occur each year. I will be able to travel to one additional conference to present, and results will be presented locally in departmental seminars. Travel to Central America to collect more samples will be coordinated and scheduled.
Impacts
What was accomplished under these goals?
Although many consumers in the U.S. may not know it, the majority of cacao we consume is grown internationally. This is because cacao is unable to be cultivated in the continental U.S. The craft chocolate industry is a fast-growing market for U.S. chocolate makers, and requires cacao beans from a variety of unique and valuable kinds of cacao. This project focuses on Criollo cacao, which is one type of chocolate prized in the craft industry. The major issues this project addresses are gaps in the understanding of Criollo cacao genetic variation, pollination, andthe impact of habitat on insects and pollinators. We are working to discover heritable variation found in wild cacao populations of fine cacao and see how agroforestry impacts pollinator abundance in the various habitats these cacao trees are found. This project is intended to provide information to farmers and resource managers about the extent of heritable variation in specific cacao types. It is also meant to provide information about which insects and pollinators are most common in different cacao habitats, which may help people optimize pollination in their local environment. Additionally, this project provides opportunities to reach students in a variety of settings and educate them on agriculture, scientific research, and the food supply chain. For each major goal in this project, the following activities, data, statistics, and discussion of results are as follows: 1. For the population genetic study of a rare, wild Criollo cacao population in Belize, leaves were collected, DNA extracted, and the whole genome sequenced. The resulting DNA sequencing data was generated and compiled with phenotypic data collected for each tree. Chloroplast DNA was extracted from the sequencing files and aligned. Preliminary population genetic analyses indicate that the population truly is Criollo cacao, which was expected. Within population variation appears to indicate two genetic clusters of trees with mixing between those two clusters. Although there is moderate genetic differentiation between those two clusters, all the trees at the station share the exact same chloroplast genome. Several possible reasons for all trees sharing the same chloroplast haplotype could be they were introduced from the same source or Criollo cacao lacks chloroplast variationmore broadly. Additionally, any historical selection for fruit traits would likely bottleneck chloroplast genomes, leading to potentially only one genotype. Further work will explore these possibilities. 2. For the study on pollinator mediated gene flow, seedlings were sprouted, collected, DNA extracted, and whole genome sequenced. Genomic DNA data was generated for 117 seedlings so far and seedlings were grouped with their "siblings" from the same pod. Additional seedlings are ready to sequence after results are analyzed for the first set of seedlings. In total,1,143 open pollinated seeds were planted, and 479 seedlings germinated and were collected for DNA extraction. This germination ratio is typical for forest trees. 3. To quantify insect variation across cacao habitats, insects were collected both manually with aspirators and passively with traps. All insects have been DNA extracted. COX1 Sanger sequences from 226 manually caught insects have been compared to the Barcode of Life Database (BOLD) for identification and have been prepared as a database to compare the passively caught bulk insects to. Trap caught insects are being prepared for COX1 metabarcoding. Results from Sanger sequencing include 226 COX1 sequences 620 bp long. Eight individuals' sequencing did not match a taxa in the BOLD Library. Successful identifications include insects from 8 orders and 38 families. The most common family was the Cecidomyiidae, or gall midges, which are a well-known presumed pollinator of cacao. Second most common was Ceratopogonidae, or biting midges, which are also a presumed pollinator. The third most common was Formicidae, or ants. Ants have recently been suggested to be significant pollinators of cacao. Discovery of three families of presumed pollinators in cacao suggests that local pollinators are common and that these taxa are likely the major pollinators in the region. Each of these pollinators have different life history strategies. Cecidomyiidae reproduce by creating galls on specific plant species. In contrast, Ceratopogonidae require a blood meal to reproduce. Knowing which pollinators may be more important in the region can help farmers facilitate appropriate habitat for pollinators. 4. Methods developed during the project so far include cacao leaf DNA extractions, which required careful troubleshooting to acquire clean DNA. Several kits and methods were investigated and both the QIAcube HT plant DNA extraction kit and Omega Bio-Tek E.Z.N.A. Plant DNA kit were successful for DNA extractions. Insect collection methods were developed by the Moulton Lab and were optimized for bulk extractions. Insect field collection methods are being compared to determine which tools reliably capture presumed pollinators. For individual insects collected near flowers, manual aspiration of insects resulted in more counts of presumed pollinators (from the families Ceratopogonidae, Cecidomyiidae, and Formicidae) when compared to sticky traps placed in flower cushions. Most insects captured with sticky traps were from Drosophilidae, which isn't known to pollinate cacao. This suggests that sticky traps placed in flower cushions are not an adequate method for studying pollinators, but manual aspiration is a successful method for capturing pollinators. Significant progress was made towards generating a finalized whole-genome sequencing data of the standing cacao at the site. Results are preliminary and will be finalized and published within the coming year. Additionally, all insects have been extracted for DNA and are ready for COX1 metabarcoding. Preliminary results provided by individually trapped insects provide insights into pollinator abundance at the research station.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
H Brabazon, JA Marlin, L Ash, E Tzalam, E Ac, M Canti, M Staton, D Shoemaker. (2025). Genomic diversity of a subspontaneous population of cacao in Belize. Evolution Conference. Athens, Georgia.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
H Brabazon, JA Marlin, L Ash, E Tzalam, E Ac, M Canti, M Staton, D Shoemaker. (2025). Genomic diversity of a subspontaneous Criollo cacao population in Belize. Botany Conference. Palm Springs, California.
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