Performing Department
(N/A)
Non Technical Summary
Chocolate, a favorite food loved by many people worldwide, is more than just an indulgence for the 50 million people worldwide who depend on it for income. Unfortunately, chocolate has a "dark side," with the average cacao farmer living well below the poverty line. Farmers' limited resources often lead to poor farming practices, which in turn lead to broader issues such as deteriorated local ecosystems and poorly managed genetic diversity in cultivated cacao trees. These problems also increase the likelihood that devastating crop diseases and climate change will negatively impact farmers' livelihoods. Fortunately, we don't need to choose between chocolate and the environment because cacao cultivation can be uniquely capable of supporting both resilient cacao production and biodiversity. My project will explore underutilized genetic diversity of cacao and shed light on cacao pollination to help farmers grow robust and valuable cacao with fewer costly inputs.I will sequence the genomes of wild cacao trees from a population in Belize to survey the genetic diversity found in a wild cacao population. These trees are highly valued for their fine flavor but are rare in cultivation. Analyses of the tree genomes will reveal which trees are pollinating each other, which will reveal how insects are moving pollen in a wild cacao forest. Lastly, I will compare insect abundance between wild and cultivated cacao trees, with a special focus on identifying potential cacao pollinators and their preferred habitats. A better understanding of the behavior of pollinators will guide farmers in accessing and improving ecosystem services to increase pollination, and thus pod production, while limiting negative effects on biodiversity.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
My long-term, overarching goal is to preserve valuable genetic diversity within cacao and minimize the impacts of cacao production on biodiversity. To accomplish this goal, it is imperative to discover and characterize natural genetic diversity maintained in wild cacao populations and understand the impact of cacao agroforestry on biodiversity and crucial ecosystem services, such as pollination.Objectives:Population genetic study of a rare, wild Criollo cacao population in Belize.Estimate gene flow, pollen transport, and pollinator movement in a wild cacao population.Quantify differences in insect/pollinator diversity between agroforestry-grown cacao and forest cacao.Develop research methods and protocols.
Project Methods
Obj. 1:To study the evolutionary history of the Criollo cacao population at BFREE, the entire 1,153-acre property was searched for cacao trees and leaves of 280 out of 291 trees were sampled for DNA sequencing. We will sample from off-site nature reserves to place the BFREE trees into the greater context of cacao genetic variation and population structure in Belize. Although a large cacao SNP chip is available, a.k.a. the "chocolate chip", expected low genetic diversity in Criollo make it likely that this SNP chip will not produce enough useful, variable markers. To sample enough markers, a novel method of quantitative reduced representation DNA sequencing, OmeSeq-qRRS, will be used to generate a dataset of thousands of markers. Preliminary data collected using this method demonstrates it captures sufficient genetic variation to distinguish between closely related trees at BFREE. Leaf DNA extraction and sequencing will follow previously optimized protocols. SNPs will be used to determine population structure by PCAand STRUCTURE. Estimates of linkage disequilibrium (non-random association of alleles), Wright'sF-statistics (allele frequency distribution), and expected vs. observed heterozygositywill be used to determine demographic history, selection levels, and inbreeding levels. All data will be analyzed in R.Obj. 2:Seeds from 1,143 open-pollinated crosses of forest trees were germinated resulting in 471 collected seedlings. Leaves will be extracted and sequenced following the same method as Objective 1. SEQUOIA parentage analysis software in Rwill identify putative relationships and parent-offspring pairs. Distances between parents will be plotted to visualize the distribution of pollination distances. The preliminary finding that Criollo at BFREE is a single, panmictic population makes it ideal for the proposed parentage analyses, because the patterns of gene flow within this population will better represent how insect pollinators move through the forest and not be biased by biological barriers to reproduction. This study will be the first to use SNP markers for a cacao parentage study.Obj. 3:Traps that are advantageous for collecting different insect families will be placed in cacao agroforest and placed in undisturbed forest near wild cacao trees. These include SLAM (Sea Land and Air Malaise), yellow pan, and sticky traps. To identify insects associated with flowers, aspirators will be used to capture flying insects on/near cacao flowers, and sticky traps will be placed next to flower cushions. Whole flowers will be collected in DNA preservative buffer for sequencing environmental or eDNA left by floral visitors. Insects will be visually sorted to order/family level (depending on taxa), and bulk genomic DNA from each set of sorted insects will be extracted. Amplification of the COX1 gene will use arthropod-specific primers and each reaction will be uniquely barcoded for next-generation MiSeq sequencing (ca. 44-50 million paired-end reads, ~500,000 reads/sample). Insects aspirated from flowers or collected on sticky traps will be sequenced individually to create a database of COX1 sequences linked to specific floral visitors at the site. Before extraction, insects collected on sticky traps will be visually inspected under a microscope for pollen, and if pollen is detected, will be sequenced for cacao DNA sequences. If cacao DNA is detected, the insect will be identified as a potential pollinator. eDNA sequencing of flowers is a novel method that has never been used for detection of cacao pollinators, but it has potential to easily screen forests and orchards for the presence of pollinators. Several extraction methods will be compared for best amplification of arthropod eDNA. Diversity indices (Shannon diversity index and Beta diversity) will be calculated within and among habitats.