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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

2022 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
Progress is being achieved during FY2022 on all three objectives as well as their sub-objectives. Under sub-objective 1a, artificial inoculation protocols and phenotyping procedures were developed, standardized, and used to complete the screening of elite Ecuadorian clones for resistance to black pod rot and Phytophthora canker. P. megakarya produces appressoria, fungi cells that allow infections to enter more readily. In contrast, P. palmivora does not typically have these cells and these pods do not develop lesions after artificial inoculation. To overcome this, high levels of the pathogens were used, and all samples were infected. Approximately 50 accessions (625 pods) from our local collection have been screened for black pod rot. In addition, over 229 accessions (915 pods) from the USDA-ARS collection in Mayaguez, Puerto Rico, have also been screened in Miami using a modified screening protocol developed in collaboration with Cacao Curator in Mayaguez. Screening for canker resistance of selected Ecuadorian clones is being carried out as part of objective 1B. Mycelia is grown in an agar petri dish and a small punch is removed. This agar infected punch is placed over a small wound manually made in the cacao branch and the infected area is measured 5 weeks later. A total of 29 accessions (86 trees) have been screened for Phytophthora canker in Miami, Florida. The goal is to be able to determine cacao’s resistance to Phytophthora canker and then perform association mapping methodology to find SNP markers linked to Phytophthora canker resistance. This information will be used in our cacao breeding program. Since the early 1930s, Badnaviruses have been severely decreasing cacao production in West Africa. Mealybugs are well known vectors of these viruses. Also, during the same time in the Americas, Trinidad was the only location where Badnaviruses were found producing losses in cacao. However, the badnaviruses, Cacao Mild Mosaic Virus and Cacao Yellow Vein-Banding Virus (CaMMV and CYVBV) have been recently found and identified in Puerto Rico, Brazil, Florida and at the International Cocoa Quarantine Centre-Reading in Reading, United Kingdom. In collaboration with scientists from the University of Arizona, ARS researchers in Miami, Florida, have developed genomic tools for virus detection and to investigate genomic variability and genetic diversity of CaMMV and CYVBV from samples that have been collected at the ARS Miami and Puerto Rico locations. These diagnostic tools are needed to accurately identify the presence of these viruses in the collection as well as in cacao farms in PR and HI. Genome sequencing and phylogenetic analysis results have shown variability in CaMMV clades among locations. The sequencing information has been used to increase the precision of new diagnostic tools to identify the presence of the viruses. These results have pointed out that Badnaviruses are prevalent in the cacao growing regions of the Americas and that the use of quarantine facilities should go hand in hand with molecular diagnostic tools as a requisite to ensure safe movement of cacao germplasm among producing countries. Because of the discovery of these viruses in the Miami quarantine facilities, the transferring of the Ecuadorian clones to Colombia, Trinidad, and Brazil as part of subobjective 1C and 1D was suspended. Regarding subobjective 2A, a total of 98,179 single nucleotide polymorphism (SNP) markers were developed using exome capture methodology using the cacao variety Matina 1-6 V1.1 assembly version and its sequence information was updated using the V 2.1 version of the Matina 1-6 genome assembly. However, a new SNP filtering process and variant calling is being conducted using the Galaxy bioinformatic tools that is available from SciNet. These SNPs will be used to screen segregating populations and identify SNPs associated with disease resistance, quality, and horticultural traits. Also, two newly designed SNP panels containing a total of 10,633 SNP markers were validated using a random set of DNA samples. The SNPs were originally designed using Illumina chips technology by the MARS and USDA-ARS collaborative project. The main objective of these two SNP development projects is to select markers that could be used in populations structure determination, identification of population admixture levels and in the discovery of markers associated with disease resistance genes and other important horticultural and quality traits. This is going to allow the development of cacao varieties using genomic-assisted breeding methodologies. Regarding objective 3, field data collection is ongoing in trials in Hawaii and Puerto Rico. In addition, segregating populations have been planted in Mayaguez, Puerto Rico, in 2021. The populations were developed from crosses made by ARS researchers in Mayaguez, Puerto Rico, in 2020 using high-yielding and disease-tolerant parents selected based on preliminary yield trial data. Leaf collection, DNA extraction and SNP fingerprinting are currently in process.

1. Selection of SNP markers core set for cacao population identification. Cacao (Theobroma cacao L.) is a worldwide, valuable tropical crop that produces the raw material used in the manufacturing of chocolate. Mislabeling cacao accessions have been a recurring problem in cacao germplasm collections around the world. Molecular markers have been widely used to address this problem; however, reference genotypes representing each one of the genetic groups are urgently needed. In addition, a set of core molecular markers that can separate the established genetic groups and identify the level of admixture in cacao accessions is also needed. Four hundred and twenty cacao leaf samples, representing all 10 genetic groups, were collected by ARS researchers in Miami, Florida, from different germplasm collections and from locations in the center of origin and domestication of cacao. Genetic characterization of the samples was done using 1,060 single nucleotide polymorphism (SNP) molecular markers. Results indicated a core set panel of 219 SNP markers that would be effective for germplasm identification and selection of reference genotypes. These results were also highly correlated with previously obtained outcomes using simple sequence repeats markers. The resulting SNP panel and the chosen reference germplasm are appropriate for cacao germplasm preservation, identification as well as their application in breeding programs.

2. Effect of cacao black pod rot screening method on disease resistance determination. Black pod rot, caused by oomycetes in the Phytophthora genus, is a devastating disease of Theobroma cacao. It is responsible for the greatest production losses of cacao around the world and resistance to this disease is a priority for breeding programs. Currently, host resistance to the pathogen is determined using the proportion of diseased pods present in the field and the size of lesions following artificial inoculation. ARS researchers in Miami, Florida, examined whether these screening methods produced different conclusions and found that they did. The resistance ranking of clones differed between screening assays. Results of this study will be used to identify new sources of resistance among accessions in the USDA-ARS Cacao germplasm collection, using both field evaluation and artificial inoculation. This improved understanding of the how screening methods used can affect the disease resistance determination in cacao germplasm will benefit breeders and farmers. Thorough characterization of cacao germplasm will enable the identification of new sources of disease resistance and more effective, long-term disease control.

3. Detection of cacao mild mosaic virus (CaMMV) using nested PCR. Cacao mild mosaic virus (CaMMV) was discovered in Trinidad in 1943 but was recently detected in Brazil and Puerto Rico. It is thought to be widespread in the Americas and spread through the movement of infected budwood. To prevent the introduction of new diseases, improved germplasm of Theobroma cacao is distributed through a quarantine system, partly located at the USDA-ARS Subtropical Horticulture Research Station in Miami Florida. In 2020, virus symptoms were observed on plants in the quarantine greenhouse in Florida during the containment period. However, the existing diagnostic test was not detecting virus, even in severely symptomatic plants. To address this, a new diagnostic test was developed by ARS researchers in Miami, Florida, and is is capable of detecting genetically diverse strains of CaMMV, and at an earlier point in the infection cycle (when virus levels are still low). In addition, a study done on dissected leaves showed that the virus in unevenly distributed, and that petiole tissue should be used for testing. This research will be used by scientists to prevent the movement and propagation of virus-infected cacao plants.

4. Molecular identification of mealybug species associated with cacao mild mosaic virus in cacao. Worldwide Theobroma cacao is affected by viruses where it is grown commercially. Most are transmitted by several mealybug species. Mealybugs belong to a diverse group known as Pseudococcidae, and each geographic region has different species composition. Cacao mild mosaic virus (CaMMV) was discovered in Trinidad in 1943, where it was shown to be transmitted by five mealybug species. It was recently detected in Puerto Rico, Brazil, and the USA; however, no information is available on insect vectors in these locations. Due to the specificity of commonly used biological control organisms (ex: parasitoids), effective management of mealybugs requires accurately identifying the species present. To enable more rapid identification of these important insects, ARS researchers in Miami, Florida, collaborated with MARS, Inc. and conducted two studies. In the first study, they developed a molecular approach that can be completed in a few days. This study compares the usefulness of the COI, ITS2, and 28S gene regions using various primer pairs to identify mealybugs, without the need for specialized taxonomic skills. Using the previously described identification tools, a second study was performed on infected trees in Florida and four species of mealybug were identified: Pseudococcus jackbeardsleyi, Maconellicoccus hirsutus, Pseudococcus comstocki, and Ferrisia virgata. Of these, P. jackbeardsleyi and M. hirsutus have not been tested for their ability to transmit viruses to cacao. CaMMV was detected in 34.6 to 43.1% of the insects tested, however, acquisition of the virus did not differ among species. This research improves our understanding of the mealybugs associated with CaMMV-infected plants and identifies potential new insect vectors. Knowledge of vector species is essential for selecting the most effective control strategies and minimizing disease spread.

Review Publications
Schmidt, J., Duval, A., Puig, A.S., Tempeleu, A., Crow, T. 2021. Interactive and dynamic effects of rootstock and rhizobiome on scion nutrition in cacao seedlings. Frontiers in Agronomy. 3:754646.
Puig, A.S., Umaharan, P. 2021. Cacao mild mosaic virus (CaMMV) and cacao yellow vein banding virus (CYVBV): genus Badnavirus. In End, M.J., Daymond, A.J., Hadley, P., editors. Technical guidelines for the safe movement of cacao germplasm. Revised from the FAO/IPGRI Technical Guidelines No. 20 (Fourth Update). Rome, Italy: Global Cacao Genetic Resources Network (CacaoNet), Bioversity International. p, 27-33.
Puig, A.S., Wurzel, S.B., Suarez, S., Marelli, J., Niogret, J. 2021. Mealybug species (Hemiptera: Pseudococcidae) associated with Cacao mild mosaic virus and evidence of virus acquisition. Insects. 12(11):994.
Gutierrez, O.A., Martinez, K.A., Zhang, D., Livingstone, D.S., Turnbull, C.J., Motamayor, J.C. 2021. Selecting SNP markers reflecting population origin for cacao (Theobroma Cacao L.) germplasm identification. Beverage Plant Research. 1: Article 15.
Puig, A.S., Irish, B.M., Ayala Silva, T., Wurzel, S.B., Gutierrez, O.A. 2022. Effect of cacao black pod rot screening method on disease resistance determination. Chemistry Proceedings. 10(1):71.