Location: Sustainable Perennial Crops Laboratory
Project Number: 8042-21000-303-001-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 14, 2018
End Date: Sep 13, 2023
Objective:
This project will address the critical need in understanding functional diversity in cacao germplasm and identify likely sources of disease resistance, new genes or alleles for breeders and an improved understanding of the disease process during the witches’ broom disease interaction. So far the efforts in characterization and evaluation of germplasm have received much less attention than germplasm maintenance. Most of the phenotyping in this collection has been limited in morphological descriptors. Only a small fraction of the germplasm held in the international collections has been evaluated for major agronomic traits and disease resistance. Within the genebank holdings, there remains a significant amount of untapped wild germplasm, which may harbor new sources of variations in resistance to diseases and pests, for environmental adaptation, and for processing quality. Through this project, we would like to evaluate the major agronomic traits, such as yield components and diseases resistance in the untapped wild populations and, analyze the susceptible and resistance mechanisms in a specific set of clones. With improved phenotyping results and high through-put genotyping using next generation sequencing and functional gene markers, we can better understand and document the functional diversity in this collection. Results of this project will contribute to more efficient management and better use of cacao germplasm for varietal development through the identification of new sources and higher levels of resistance to cacao diseases and other important agricultural traits. The new disease resistance genes identified can be used by cacao breeders to improve breeding programs. The objectives of the agreement directly relate to specific project objectives in the NP301 project “Characterizing and Evaluating the Genetic Diversity and Horticultural Value of Genetic Resources for Cacao and Other Tropical Tree Crops Economically Important to the United States and in NP303 project “Developing Pathogen-and Plant-Based Genetic Tools for Breeding Disease Tolerance in Theobroma cacao.”
Approach:
Genetic diversity and phenotypic trait analysis of the International Cocoa Genebank, Trinidad (ICG,T). A subset of 200-300 cacao germplasm accessions held in CRU/ UWI (approximately 10% of the entire collection) comprising different wild cacao populations will be selected. The criteria to assemble this working collection will be based on (i) confirmed genetic identity through previous DNA fingerprinting, ii) geographic origin and genotypic diversity, and (iii) historical importance of the accessions. Phenotyping will be carried out in the International Cocoa Genebank, Trinidad. Within plot (intra-clone) mislabeling will be identified using 40-50 SNPs. Phenotypic traits related to yield components and diseases resistance will be evaluated, including number of pods, pod index, bean number, bean size, bean weight, butter fat content, and field resistance to witches’ broom disease and black pod. Previously recorded data on morphological variations, (Bekele et al., 2006) will be combined with new observations. Where duplicated trees in different environments are available, phenotypic data will be recorded from different environments. Technology of next generation sequencing and candidate gene markers will be used to genotype these wild populations. Population structure patterns will be investigated using the Bayesian clustering method implemented in the STRUCTURE program. Both single marker models and haplotype-based tests will be applied for all SNP–trait combinations. Marker data will be compared with trait values by three statistical methods. Patterns of linkage disequilibrium (LD) among SNPs that are significantly associated with the same trait will be assessed using the maximum-likelihood approach implanted in GENETICS.
Analysis of cacao meristem reaction to the Moniliophthora perniciosa pathogen.
Utilizing RNAseq technology, cacao clones with varying levels of resistance to the fungal pathogen that caused witches’ broom disease, will be infected. Plants will be produced from 8 clones to develop a replicated 2 time-point experiment with 5 reps/time point/clone-plus controls. A total of 20 plants per clone will be produced for a total of 160 samples. The clones will be inoculated with M. perniciosa and the infected material will be harvested at the 2 time points, representing one early and late time point in the disease cycle. The material will be frozen in Liquid nitrogen and kept at -80C. The RNA will be extracted from 3 reps/time point/clone plus controls for a total of 96 samples. A decision will be made later to determine if leaves or stems are the most useful material based on RNA extractions and gene expression. Once completed the RNA (or frozen material will be shipped to Beltsville or directly to BGI for sequencing. Sequencing costs will be covered separately from the budget through another funded agreement.
