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Research Project: Development and Application of Genomic-assisted Breeding Strategies to Produce Disease-resistant Cacao Genetic Resources

Location: Subtropical Horticulture Research

2019 Annual Report

1. Evaluate (phenotype) cacao genetic resources for host-plant resistance to the priority diseases black pod, frosty pod, and witches’ broom. Record and disseminate evaluation data via the project’s website, the Cacao Genome Database, the International Cocoa Germplasm Database, and/or other data sources. 1A. Develop artificial inoculation protocols and phenotyping techniques to assess resistance to Phytophthora canker in clones from Ecuador. 1B. Assess Ecuadorian and NPGS cacao germplasm in Puerto Rico for resistance to BP and make data available via cacao databases. 1C. Screen Ecuadorian clones for FP resistance in Colombia and make data available via cacao databases. 1D. Evaluate Ecuadorian clones for resistance to WB in Colombia, Trinidad, and Brazil and make data available via cacao databases. 2. Develop and apply more effective genetic tools, including genetic markers, association mapping, genome-wide selection, and/or experimental populations, for breeding cacao genetic resources with host-plant resistance to the priority diseases black pod, frosty pod, and witches’ broom. 2A. Use exome capture methodology to develop SNP markers from diverse cacao germplasm and utilize them for screening segregating populations and germplasm exhibiting resistance to BP, FP, and WB, as well as desirable horticultural and quality traits. 2B. Utilize genome-wide selection (GWS) models to predict genomic breeding values (GBV) in full-sib family cacao crosses. 3. Breed and release cacao genetic resources with increased host-plant resistance to the priority diseases: black pod, frosty pod, and witches’ broom.

Currently, around thirty percent of the world annual cacao production is lost to pest and diseases. In the Americas, the major losses are caused by three diseases: black pod rot (BPR), caused by several different Phytophthora species of which P. palmivora is the most common, frosty pod rot (FPR), caused by Moniliophthora roreri, and witches’ broom (WB), caused by Moniliophthora perniciosa. Presently over 250 cacao germplasm accessions are available at the USDA-ARS-TARS in Mayaguez, Puerto Rico. However, most of this germplasm has not been evaluated for resistance to these diseases. Screening protocols for selecting germplasm resistance to Phytophthora canker and BPR will be used to select accessions resistant to these diseases. In addition, selected accessions from the USDA-ARS/MARS/INIAP collaborative breeding program will be sent to Colombia for evaluation to BPR, FPR and WB. Also due to the presence of different WB pathotypes in Trinidad and Brazil, these accessions will be also screened for resistance in these countries. Single Nucleotide Polymorphisms (SNPs), both as single base pair substitutions and single base pair insertions/deletions (indels) are the most common sequence differences found between alleles. Due to the sequence of two cacao genomes, Matina 1-6 (Amelonado) and B97-61/B2 (Criollo), methodologies such as whole-exome targeted sequencing have been developed and this has facilitated SNP discovery. Once SNPs have been identified, they can be employed to genotype cacao accessions previously screened for disease resistance as well as populations segregating for disease resistance. Cacao breeding is a slow process, due to the tree’s long reproductive cycle. Therefore, the development of SNP markers and the application of genomic-assisted breeding methodologies will be implemented to strengthen the effectiveness of the selection process. Since FPR and WB are not present in Hawaii and Puerto Rico as well as other cacao producing countries in the Americas, preventive breeding for these diseases will be extremely useful.

Progress Report
Objective 1a has been completed, following the inoculation of 100 seedlings (with a range of resistance levels) with P. palmivora, and comparing lesion development. Results from this were used to select the screening method best able to differentiate among different levels of resistance to Phytophthora canker. This screening method is essential for addressing Objectives 2 and 3. The grafted plants necessary to screen for canker resistance in Objective 1b are being generated and screening has begun on a subset of these using the screening protocol developed. Ecuadorian clones are also being artificially-pollinated at SHRS, and these pods are being screened for resistance to black pod rot using artificial inoculation of detached pods. Five years of disease incidence data for cacao clones in the germplasm collection in Puerto Rico is being reviewed and a subset of promising clones will be screened for resistance to black pod rot. To understand environmental factors and management techniques that may be contributing to disease development, disease incidence and weather data are being collected at a research farm in Ecuador. Two years of data have been collected and results from these epidemiological studies will be presented at the American Phytopathological Society Plant Health Meeting this August in Cleveland, Ohio. Volatiles have been characterized from healthy and diseased pods of 6 genotypes as part of a collaborative project with ARS researchers in Miami, Florida. We made progress toward demonstrating that different cacao genotypes release different compounds, which may be responsible for the differing levels of insect damage occurring in the field. Insect damage of the pod epidermis dramatically increases the likelihood of infection, and differences in attractiveness among clones makes it difficult to interpret disease incidence data collected in the field. Progress was also made in assessing pathogens present in Hawaii and Ecuador. Phytophthora spp. and other pathogens isolated from diseased pods and stems were shipped to Miami for pathogenicity tests or completion of Koch’s postulates. A total of 50 Phytophthora isolates were obtained, DNA was extracted, and they have been barcoded for diversity analyses (ITS and COX1 or COX2). High molecular weight genomic DNA was extracted from a subset of isolates and sent to University of California - Davis (UC-Davis) for genome sequencing as part of the pathogenomics project (UC Davis/Mars/USDA-ARS). These isolates comprise the majority of P. palmivora isolates included from the Americas. In addition, a new pathogen, Neofusicoccum parvum, was identified, and a series of inoculation studies was carried out to assess its severity as a pathogen and define the infection cycle. Field trials, commercial production areas, and the USDA cacao germplasm repository in Puerto Rico will be visited at the end of August to determine what diseases are present there. A delay of approval of the Material Transfer Agreement (MTA) needed to send the cacao clones to Colombia, has slowed down the process of Subobjectives 1c and 1d. It is expected that the MTA will be approved soon. In addition, conversation, for the transferring of the clones to Brazil and Trinidad, is in process. Currently, MTA drafts are being designed. Regarding Subobjective 2a, DNA was extracted from 1,054 cacao accessions that included germplasm from Ecuador, the USDA-ARS cacao germplasm collection, diverse cacao accessions from Mexico, Honduras, Puerto Rico, Trinidad as well as a random mating and a disease resistant segregating population. The DNA samples were subjected to sequencing and exome capture methodology. Preliminary results indicated that at least 97,569 Single Nucleotide Polymorphic (SNPs) markers were identified. Further in-house analysis is being conducted and validated SNP markers will be used to develop linkage maps, characterize cacao population structure, conduct Quantitative Trait Loci (QTL) discovery, association mapping and genomic selection studies. The final goal is to identify SNP markers associated with disease resistance, quality and horticultural traits that would allow the development of cacao closes using genomic-assisted breeding approaches. Concerning Objective 3, cacao yield data collection has been conducted by ARS collaborating researchers for year three in Puerto Rico and for the second year in Hawaii. Preliminary data analysis is being conducted to determine if any of the cacao clones are outstanding at an early stage of testing. It has been established that cacao has eleven different genetics groups and the estimation of the level of admixture present in each clone that will be used in a crossing scheme is highly recommended. Currently the fingerprinting analysis of selected clones is under way. Once this process is completed, the crossing of selected clones will start. Our collaboration with the University of Arizona regarding the development of diagnostic tools for the identification of badnaviruses in cacao has continued. A collection of cacao leaf samples from trees presenting symptoms of cacao swollen shoot disease (CSSD) were collected in Cote d´Ivoire and Ghana by researchers in these countries and sent to our collaborator at the University of Arizona. DNA was isolated and subjected to Illumina DNA sequencing at their facility. Genome sequences were analyzed, and 30 newly determined genomes were identified which included three badnavirus species, Cacao swollen shoot virus (CSSV), Cacao swollen shoot CD virus (CSSCDV), and Cacao red vein virus (CRVV) were characterized. Preliminary results indicated that population structure for the CSSV and Cacao red vein banding virus (CRVBV) isolates appears to be native to the region comprising Ghana and Cote d’Ivoire in West Africa. This information helps in the development of an accurate diagnostic tool for CSSD. CSSD is not present in the Americas and the international collaboration with countries in West Africa has been vital. The development of a diagnostic tool to screen germplasm during the quarantine process is of paramount importance. At the Centro Agronómico Tropical de Investigación y Enseñanza (CATIE), data collection in inoculations of the fungus that causes Black Pod Rot and Frosty Pod Rot has continued for the fifth year in the F2 mapping population of the cross ‘Pound 7’ x ‘UF 273’. This work has been done to determine the level of resistance to these diseases at the individual tree level because we need to establish an association between molecular markers and the level of the resistance to the diseases presented by a single tree. Also, DNA extraction from leaf samples has been conducted and genotyped with 97,569 SNP markers. The main objective of this research is to discover the genes associated with Black Pod Rot and Frosty Pod Rot resistance. Phenotypic and genotypic data analysis is in progress. The outcome of this research is the development of cacao disease resistant clones using a genomic-assisted approach. In Trinidad, data collection of self-incompatibility on selected cacao accessions has continued. The main purpose of this research is the identification of genes associated with self-compatibility in cacao which is in the main objective of this collaboration. These cacao clones will be fingerprinted with SNP markers and Association Mapping methodology will be applied to find associations between SNP markers and genes controlling self-compatibility.

1. Detected a previously-unreported species of Phytophthora causing canker on Theobroma cacao in Ecuador, constituting a new record for this host. Disease data collected at a research farm in Ecuador, showed that one of the plots being evaluated had no black pod rot history despite having a high incidence of Phytophthora canker. ARS researchers in Miami, Florida, used isolates collected from this plot in 2018 in Ecuador as part of a collaborative epidemiological study with Mars, Inc. researchers. A species of Phytophthora previously unreported on cacao was discovered from stem canker and soil.

2. Identified Phytophthora palmivora as the predominant species causing black pod rot in Hawaii. Black pod rot, caused by Phytophthora spp., is the only major cacao disease that has been reported in Hawaii, but distribution information and molecular identification are lacking. In order to determine the etiology and extent of Phytophthora spp. diseases on Theobroma cacao, ARS researchers in Miami, Florida, used pod samples collected from Hawaii and Oahu Islands (eight total sites). Phytophthora palmivora was found on three of the eight sites, causing both pod rot and stem canker. The pathogen was not detected on Oahu, despite the presence of symptomatic pods, and no other Phytophthora species were detected.

3. Identified a new pathogen, Neofusicoccum parvum, as responsible for a subset of the black pod rot observed on cacao in Hawaii. ARS researchers in Miami, Florida, identified a new pathogen, Neofusicoccum parvum, as being responsible for a subset of the pod rot observed on cacao in Hawaii, as it causes symptoms identical to those caused by Phytophthora spp. This highlights the importance of confirming pathogen identity via isolation before attributing field symptoms to P. palmivora.

4. Developed a screening method to assess resistance to Phytophthora canker in cacao. Various artificial inoculation methods were conducted to determine the infection cycle of Phytophthora spp. on cacao. Preliminary results show that stem infection can occur in the absence of wounds, leading to development of stem cankers. This infection route had not been previously reported in scientific literature. Of the five methods tested, inoculation with mycelial plugs following wounding with a needle, was more reliable than using zoospores. It uses 1-2-year-old seedlings in growth chambers and provides greater resolution than screening using detached stems. This screening procedure used by ARS researchers in Miami, Florida, provides accurate phenotypic information concerning the level of resistance to Phytophthora canker by each one of these accessions. This information will be used to find association between single nucleotide polymorphic markers and the level of resistance to Phytophthora canker. The resulting linked markers will be used in our genomics-assisted breeding program.

Review Publications
Cornejo, O.E., Yee, M., Domingez, V., Andrews, M., Sockell, A., Strandberg, E., Livingstone, D., Stack, C., Romero, A., Umaharan, P., Royaert, S., Tawari, N.R., Ng, P., Gutierrez, O.A., Phillips, W., Mockaitis, K., Bustamante, C.D., Motamayor, J.C. 2018. Population genomic analyses of the chocolate tree, Theobroma cacao L., provide insights into its domestication process. Communications Biology. 1:16.