Location: Subtropical Horticulture Research
2024 Annual Report
Objectives
1: Breed and release novel cacao germplasm and cultivars with superior yield, host-plant resistance to diseases, and/or tolerance to drought and elevated temperatures.
2: Conduct research to identify genetic markers and develop integrated genetic maps to support cacao breeding for resistance to biotic stresses and tolerance of environmental extremes.
2.A: Identify association between SNP markers and resistance or tolerance to Badnaviruses, Poleroviruses, Lasiodiplodia spp. Colletotrichum spp., Diaporthe spp and Neofusicoccum parvum in cacao germplasm.
2.B: QTL discovery for resistance to BPR in six full sib families containing two different sources of resistance and gene identification.
Approach
Currently, the major losses in cacao are due to 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. In addition, other emergent pathogens (Lasiodiplodia spp. Colletotrichum spp., Diaporthe spp and Neofusicoccum parvum) that in the past have been considered of minor importance have started to influence cacao production worldwide. Also, viruses (Badnaviruses and Poleroviruses) have been recently reported affecting cacao production in the Americas. Therefore, screening protocols for selecting germplasm resistance to these pathogens will be conducted by this project in the cacao germplasm accessions and in segregating populations at the USDA-ARS-TARS in Mayaguez, Puerto Rico since sources of resistance must be found. Even though, FPR, and WB are currently not found in Puerto Rico and Hawaii. Nonetheless, this project will conduct a preemptive breeding strategy based on evaluating cacao segregating populations developed in Puerto Rico for host-plant resistance in countries where those diseases are present to develop disease-resistant varieties before these diseases arrive in Hawaii and Puerto Rico. Several cacao accessions have been sequenced and their genome information is available. Single Nucleotide Polymorphisms (SNPs) markers have been developed using this sequence information and will be used to genotype cacao accessions and segregating populations previously screened for resistance to these diseases. Associations between SNP markers and gene for disease resistance will be pursued during this project. Since Cacao breeding is a slow process, due to the tree’s long reproductive cycle. Generated genotypic and phenotypic information will be used in combination to identify Quantitative trait loci (QTL) associate with target traits using genome-wide selection (GWS) methodology as well as genomic selection models to strengthen the effectiveness of the selection process and accelerate the development of disease resistant cultivars. This research effort deliver cacao improve germplasm and save the livelihood of thousands of small farmers in the Americas.
Progress Report
Progress was made during FY2024 on both objectives and their sub-objectives. Under Objective 1. Breed and release novel cacao germplasm and cultivars with superior yield, host-plant disease resistance, and tolerance to drought and elevated temperatures. A total of 432 trees from 6 full-sib families 1) TARS 14 × SHRS 13, 2) TARS 23 × SHRS 13, 3) TARS 23 × SHRS 15, 4) TARS 30 × SHRS 03, 5) TARS 30 × SHRS 15, and 6) TARS 31 × SHRS 15 were planted in Corozal, Puerto Rico and are being screened in Miami, Florida with 2 Single Nucleotide Polymorphic (SNP) markers chips, each one containing 5.5 K SNPs. A paternity analysis will confirm the parents' identity in each cross. Phenotypic data, including pod counts and disease incidence, was collected for the first year.
Due to the update in 2023 of the USDA Plant Hardiness Zone Map, a new approach to developing breeding populations in Miami started at the beginning of FY 2024. Four new breeding populations with a total of 183 progenies have been created. After being fingerprinted with SNP markers, these will be planted in a selected area towards the end of the fiscal year. In addition, cacao-chosen cultivars from the local collection have been cloned and planted at two locations where they will be subject to abiotic stresses such as flooding and elevated temperatures.
Objective 2. Conduct research to identify genetic markers and develop integrated genetic maps to support cacao breeding for resistance to biotic stresses and tolerance of environmental extremes. Sub-Objective 2.A: Identify the association between SNP markers and resistance or tolerance to Badnaviruses, Poleroviruses, Lasiodiplodia spp. Colletotrichum spp., Diaporthe spp and Neofusicoccum parvum in cacao germplasm. ARS scientists in Miami, Florida, and Mayaguez, Puerto Rico observed visual virus symptoms at the National Plant Germplasm System (NPGS) cacao collection. They collected leaf samples and sent them to collaborators at the University of Arizona for further testing. Preliminary results indicated that the collection is infected with CaMMV and that at least six variants are present. Samples presenting visual CYVBV symptoms were also collected and sent to collaborators at the University of Arizona, where a test to detect CYVBV was developed and is in the process of optimization. The sequence of Polerovirus samples found at the Miami Quarantine Station has been completed by collaborators at the University of Arizona, and a diagnostics assay is going to be used to identify the presence of Poleroviruses in the National Plant Germplasm System (NPGS) cacao collection and breeding populations. Phenotypic disease data for the presence of Lasiodiplodia spp. Colletotrichum spp., Diaporthe spp., and Neofusicoccum parvum are being collected in the collection, and protocols for the screening against these diseases are being developed. Accurate diagnostic tools and screening methodologies for pathogens affecting cacao production are paramount because they have been found in cacao collections and breeding programs across the Americas and affect cacao production. Therefore, their use is mandatory to ensure the safe movement of cacao germplasm among producing countries and to breed resistance against these pathogens.
Regarding Sub-Objective 2.B: QTL discovery for resistance to BPR in six full-sib families containing two different sources of resistance and gene identification, a Phytophthora spp. Isolate had been selected for the screening and preliminary tests conducted in Miami, Florida. Budwood from the six full-sib families contains two different sources of resistance to Phytophthora spp. will be sent to Miami to be planted in a selected location at the Subtropical Horticulture Research Station to facilitate the rapid Phytophthora spp. screening of these segregating populations.
Accomplishments
1. Genome sequencing of the cacao leafroll virus. Polerovirus infecting Theobroma cacao germplasm was detected at the Miami
Quarantine facilities in 2022. ARS researchers at Miami, Florida believe Cacao breeders worldwide need a safe exchange of germplasm to introduce new sources of genetic variation to their breeding program. The identification of pathogens during the quarantine process is important to avoid the introduction of diseases not previously present in these areas. Leaves with virus-like symptoms that consist of leaf discoloration and distortion with downward rolling at leaf edges and light-yellow speckling or mottling were observed on cacao grafted trees of the CCN 51, CATIE-R1, CC-137, and CATIE-R4 genotypes that were undergoing the quarantine process at the Subtropical Horticulture Research Station of the USDA-ARS in Miami, Florida.
Collected leaf samples of symptomatic plants were shipped to the University of
Arizona for further analysis. RNA was extracted from the samples, RNA sequencing
was conducted, and the results were compared with plant virus databases. The
complete genome sequence of cacao leafroll virus (CaLRV; family Solemoviridae,
genus Polerovirus) was determined by comparing its sequence to other
Poleroviruses in cotton, melon and cereals. Diagnostic tools for this Polerovirus
have been developed. They are being used to screen cacao germplasm for the
presence of this virus at the quarantine station and at the cacao collections at
USDA-ARS.
2. Genome sequencing of three wild cacao accessions. Cacao, the chocolate tree, is severely affected by diseases worldwide. ARS researchers at Miami, Florida suggest developing cultivars with disease resistance is a paramount priority for the plant breeding programs. Cacao accessions with high levels of disease resistance have been collected in the upper Amazon area and used as parents in breeding programs. However, the cacao breeding process is slow due to the tree’s long reproductive cycle. It takes more than 14 years to release a cultivar. Therefore, there is a need to speed up the development of cacao cultivars. The sequencing of the cacao genome has provided information for the development of molecular markers associated with disease resistance traits. USDA ARS scientists from Beltsville, Maryland and Miami, Florida, in collaboration with researchers from the University of Nebraska, sequenced the cacao accessions, Pound 7, IMC 67, and Scavina 6 belonging to the Nanay,Iquitos, and Contamana genetic groups, respectively. Pound 7 has been used as a source of resistance to black pod rot. IMC 67 is resistant to Ceratocystis wilt and Scavina 6 is a known source of resistance to black pod rot and witches’ broom. A de novo assembly approach with HiFi long reads sequencing was scaffolded using a reference-free strategy. Recently the importance of de novo sequencing of multiple reference genomes to obtain the genome diversity has been pointed out. These genome assemblies are part of the three most valuable genetic clusters of cacao accessions from the Upper Amazon region. The three wild cacao genomes offer important resources for accelerating the development of disease resistant cacao cultivars.
3. Development of Cacao Breeding Populations in Miami. Cacao is a tropical perennial crop that usually grows 20 degrees north and south of the equator. However, recent changes in the 2023 USDA Plant Hardiness Zone Mapping located Miami, Florida in the 11a zone, and would allow the planting of cacao breeding populations in the area. Four segregating populations with a total of 183 progenies have been obtained and will be planted in selected areas at the end of FY 24. SNP markers are being used to confirm the identity of the parents. Furthermore, cacao clones from the station collection are being cloned and an experiment to evaluate abiotic stresses such as flooding and elevated temperatures will be planted at two different locations in the Miami area.