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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Sustainable Perennial Crops Laboratory » Research » Research Project #434392

Research 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

Location: Sustainable Perennial Crops Laboratory

2020 Annual Report

Objective 1: With NPGS and international cooperators, elucidate the geospatial patterns of genetic diversity for the primary gene pool of cacao; strategically acquire cacao genetic resources to fill gaps in NPGS and other genebank collections; and incorporate genetic diversity data into this project’s website, international cacao genetic resources databases, and GRIN-Global. [NP301, C2, PS2A; C4, PS4] Sub-Objective 1A: Elucidate geospatial patterns of genetic diversity for the primary gene pool of cacao. Sub-Objective 1B: Assess whether the genetic diversity in ex situ collections is representative of cacao’s primary gene pool. Fill genetic gaps in those collections by strategically collecting new accessions from natural populations and farmer fields. Sub-Objective 1C: Incorporate genetic diversity data into the project’s website, international cacao genetic resources databases and GRIN-Global. Objective 2: With domestic and international cooperators, characterize and evaluate cacao genetic resources for tolerance to abiotic stresses, for adaptation to growth under different environments and horticultural management regimens, and for other priority horticultural traits. [NP301, C2, PS2A; C1, PS1A] Sub-Objective 2A: Evaluate cacao germplasm for tolerance of soil moisture deficits to identify tolerant clones for breeding drought-tolerant varieties. Sub-Objective 2B: Evaluate cacao germplasm for accumulation and translocation of heavy metals, such as cadmium; assess nutrient use efficiency in different environments to identify superior clones for breeding varieties with high nutrient use efficiency and low concentration of toxic heavy metals. Objective 3: Develop and apply genomic tools for improving the efficiency and effectiveness of managing and utilizing genetic resources of other priority tropical crops, such as tea, guava, longan, rambutan, pitaya, star fruit, mangosteen, peach palm and macadamia nut. [NP301, C2, PS2A] With support from this budgetary increase, more effective coffee genetic resource evaluation, and characterization methods will be developed and applied, focusing on germplasm to be incorporated into the new USDA/ARS coffee genetic resource collection.

Firstly, the project will elucidate geospatial patterns of genetic diversity in the primary gene pool of Theobroma cacao using genomics, spatial genetics and bioinformatics. Wild cacao trees originated from Colombia, Peru, Ecuador, Bolivia, Brazil and French Guiana will be genotyped using Next Generation Sequencing. The NGS data, in conjunction with GIS and ecological information, will be analyzed to reveal distribution of Theobroma cacao in the Amazon. The resulting information will serve as a scientific baseline to support rational decision-making for future germplasm conservation and utilization. Diversity gaps in ex situ collections will be identified and filled through new collection expeditions to increase representation from the geographical centers of diversity and collect landraces and traditional varieties to support in situ/on-farm conservation. In collaboration with USDA’s GRIN-Global team and international partners, this project will also improve the genetic integrity of the genebank holdings and allow us to significantly improve the accuracy of information in the public databases. Secondly, this project will evaluate cacao germplasm for tolerance to key abiotic stresses and horticultural traits, with the emphasis on drought tolerance and lower uptake and transport of Cd to improve the productivity and quality of cacao beans. Research will be conducted with research institutes and universities in Peru, Brazil, Puerto Rico and Ecuador. Cacao genetic resources will be characterized and evaluated in different agricultural ecologies in the Americas for tolerance to abiotic stresses. Field studies will be implemented with drought-tolerant genotypes identified to assess their growth performance and yield potentials under different cacao growing regions of South America. Interntional germplasm will be evaluated to identify genotypes that are tolerant to toxic levels of Cd. New parental genotypes with superior ability to establish under abiotic stresses and superior tolerance to drought and low uptake of soil Cd conditions, will be incorporated in breeding programs. A third objective is to assess the diversity of other less studied tropical fruit and nut species. Genomic tools will be developed for improving the efficiency and effectiveness of managing and utilizing genetic resources of tropical fruits and nuts, such as tea, coffee, guava, longan, rambutan, pitaya, star fruit, mangosteen, peach palm and macadamia. SNP markers will be developed through data mining and/or NGS technology for these species. These putative SNP markers will be validated and evaluated for suitability in germplasm identification and genetic diversity assessment. High quality SNPs will be selected to form a genotyping panel for each species, which will be applied for SNP fingerpritting of germplasm of tropical specialty crops maintained USDA tropical fruits and nuts germplasm collections in Hilo, Hawaii and Mayagüez, Puerto Rico.

Progress Report
Progress was made under Sub-Objective 1A: In collaboration with scientists from the Colombian Agricultural Research Corporation (AGROSAVIA), a pilot study was carried out to characterize farmer selections of cacao in Colombia using single nucleotide polymorphism (SNP) markers. The analytical result revealed that these farmer selections were mainly hybrids between traditional Trinitario and a small number of introduced Upper Amazon Forasteros (UAF). This result demonstrates a narrow genetic background in farmer selections in Colombia and suggests that diverse UAF germplasm, including those indigenous to Colombia, need to be utilized in the breeding program of AGROSAVIA to improve the tolerance to biotic and abiotic stresses and other agronomic traits. Progress was made under Sub-Objective 1B: In collaboration with scientists from the Colombian Agricultural Research Corporation (AGROSAVIA), cacao germplasm previously collected from the Amazonian Department were analyzed using SNP markers. The result demonstrates significant sub-structure among different river systems in the Colombia Amazon, suggesting the needs for additional collecting expeditions in these rivers, including Caqueta, Vaupes, Yari, Apaporis, Negro and Putumayo. Under Sub-Objective 1C, progress was made in verification of two cacao working collections housed at Pennsylvania State University and at the Central Agricultural Research Institute (CARI), in Suakoko, Liberia. The verification led to the correction of mislabeled clones and falsely recorded pedigrees thus improved the integrity of the collections. The genotyping data, as well as the analytical results were submitted to the International Cacao Germplasm Database (ICGD), Reading, U.K. Considerable progress was made under Sub-objective 2A, in collaboration with scientists from Cacao Research Institute (CEPLAC/CEPEC) Bahia, Brazil, and the Tropical Crop Research Institute (ICT) in Tarapoto, Peru in assessing abiotic stresses (drought, soil acidity, light quality) of national and international cacao varieties and genotypes collected from various Peruvian river basins. In Peru, research to evaluate effects of solar radiation (50% and 20% full sunlight) on the growth, physiological and nutrient use efficiency of 60 wild and domesticated cacao genotypes revealed interspecific differences for plant height, stem diameter, biomass and chlorophyll content. The diversity identified could be exploited in breeding programs to develop cultivars with increase tolerance to shade. In another study 60 wild and domesticated cacao genotypes were evaluated for growth, physiological and nutrient use efficiency at 0 and 30% soil aluminum saturation and the obtained data is being analyzed. In Brazil experiments were conducted to evaluate relationships between mineral nutrient ratios of cacao leaf to cacao productivity from 48 cacao cropping systems. In another study the interactions between soil, leaves and bean nutrient status and dry biomass of beans and pod husk were evaluated. The nitrogen and potassium contents of cacao leaves were directly related to high dry biomass of cacao beans. The levels of exchangeable calcium and magnesium in the soil were inversely correlated with the dry biomass of cacao pod husk and the micronutrients zinc, manganese, iron and copper in leaves were positively associated with the dry biomass of cacao pod husks. Under Sub-objective 2B considerable progress was made in collaboration with scientists from Cacao Research Institute (CEPLAC/CEPEC) Bahia, Brazil, Tropical Crop Research Institute (ICT) in Tarapoto, Peru was made in the mitigation of cadmium (Cd) toxicity in cacao and in the evaluation of soil amendments for reducing bioavailable of Cd. ARS scientists at Beltsville, Maryland, completed experiments to evaluate the growth and physiological response of cacao genotypes to toxic levels of soil cadmium. The resulting plant samples are being analyzed. In Peru, research was undertaken to assess the effects of soil amendments such as calcium hydroxide, dolomite, island guano, cocoa husk compost, and chicken manure, triple superphosphate, di-ammonium phosphate, potassium chloride and potassium sulfate on the bioavailability of mitigation cadmium in the soil. Data were compiled on the uptake and growth variations of the cacao genotype CCN51. In Peru, experiments to evaluate the growth and nutritional responses of 60 wild and domesticated cacao genotypes to soil cadmium stress has been completed. Intraspecific differences were observed for growth, nutrient uptake and nutrient use efficiency and the accumulation of Cd in soils with toxic levels of soil Cd. In Brazil, the role of manganese was evaluated for its ability to mitigation cadmium and lead toxicity effects on physiological, biochemical, molecular, micromorphological and ultrastructural changes in young cacao clones. Increased manganese concentration in the soil decreased cadmium uptake by the roots and reduced the transport of cadmium to the leaves. In another study, the foliar applied of a cuprous oxide fungicide was evaluated for its effect on the physiological, biochemical and molecular changes in cacao leaves and was found to affect the growth and development of cacao leaves. Significant progress was made under Objective 3. A single nucleotide polymorphism (SNP) genotyping panel for identification of Arabica coffee (C. Arabica) was developed. This genotyping panel, comprised of 96 SNP markers, was tested on 288 Arabica varieties sampled from Brazil, Costa Rico, Puerto Rico and Cote d'Ivoire. All tested varieties were unambiguously identified. Mislabeled genotypes and falsely recorded pedigrees were accurately detected. This genotyping panel is now being used to characterize all Arabica coffee accessions maintained in the International Coffee Genebank, hosted in the Tropical Agricultural Research and Higher Education Center (CATIE), Costa Rica. The panel is also being used by the World Coffee Research (WCR) to certify coffee plant nurseries in Latin America and Africa for producing healthy and true to type planting materials. Under the Objective 3, significant progress was made in application of SNP markers to assess genetic diversity in Robusta coffee germplasm. Using the SNP genotyping panel developed by ARS scientists in 2019, a total of 2,100 Robusta coffee accessions from the national coffee collections of Ghana, Kenya and Cote d'Ivoire were genotyped. The results enabled the detection of significant population structure and high rate of mislabeling in the collection, demonstrating the significant implication to improve the accuracy and efficiency in Robusta coffee germplasm management. Additional progress was made in assessment of genetic diversity in wild jujube (Ziziphus jujube var. spinosa) populations. Wild jujube is the ancestor of cultivated jujube and is indigenous to China, where it is utilized in the value chain of horticulture, food and traditional medicine. In collaboration with Ningxia University in China, scientists from USDA, ARS in Beltsville, Maryland, analyzed the genetic diversity in five wild jujube populations distributed in Ningxia, China, using the approach of genotyping by sequencing (GBS). Significant geographic division were detected, which coincides with the current classification of major agroecological zones in these regions. Within each region, there is significant population differentiation, which was explained by the isolated habitat and limited gene flow among the populations. The results illustrate the impact of natural habitat fragmentation for wild jujube in the arid and semi-arid areas, which provided useful information for conservation and using this vast untapped genetic diversity for breeding jujube with improved adaptability to abiotic stresses. Under the same objective significant progress was made in collaboration with scientists from the Kearney Agricultural Research Extension Center (KAREC), Division of Agriculture and Natural Resources, University of California, Davis, California; the genetic diversity of the California tea germplasm collection was analyzed. Tea is a specialty crop with great potential for smallholder farmers in California. The California tea germplasm were analyzed together with reference populations from China, India and Southeast Asia using SNP markers developed by ARS scientists at Beltsville, Maryland. The results revealed the genetic background of the California tea germplasm accessions, some of which have unique identity with a hybrid genetic make-up of both Camellia sinensis and C. assamica. Finally, under this objective, in collaboration with Dovetail Genomics, LLC (Scotts Valley, California) and the University of Nebraska, Lincoln, developed the first draft genome of rambutan (Nephelium lappaceum). The 409.23 Mbp genome of rambutan was sequenced using long-read sequencing and two proximity ligation methods. It is estimated that over 99% of the rambutan genome was assembled into 979 scaffolds. The draft genome provides a high-quality reference for SNP discovery, diversity analysis and crop improvement for this important tropical fruit from the Sapindaceae family.

1. Development of a core set of Single Nucleotide Polymorphic (SNP) markers for genotyping Arabica coffee. Accuracy and efficiency are essential for coffee germplasm management, and for the exchange and utilization of these genetic materials in breeding new varieties of coffee with superior traits. A universal genotyping panel that allows data comparison across different laboratories and genotyping platforms was not available for Arabica coffee (Coffea arabica). Using Nano-Fluidic Array genotyping technology, ARS scientists from Beltsville, Maryland, validated 800 candidate SNP markers on Arabica coffee accessions obtained from Brazil, Costa Rico, Puerto Rico, and Cote d'Ivoire, through research collaborations. The final genotyping panel, comprised of 96 high quality SNP markers, was capable of unambiguously differentiating all tested varieties of Arabica coffee, despite their high genetic similarity and accurately identified mislabeled accessions. This panel is also being used by the World Coffee Research (WCR) to certify coffee nurseries in Latin America and Africa for producing healthy true-to-type planting materials for farmers. This information will be useful to coffee breeders, germplasm curators, seed producers, coffee growers, and the coffee industry.

2. The role of manganese in the mitigation of cadmium toxicity in young cacao plants. In South America, soils under cacao invariably have high levels of cadmium. Cacao tends to absorb high level of cadmium which is toxic to cacao and inhibits growth and quality of cocoa beans, leading to reduced quality and detrimental impacts to chocolate makers and consumers. ARS scientists from Beltsville, Maryland, with scientists from Cacao Research Institute (CEPLAC/CEPEC) Bahia, Brazil found that increased levels of manganese could moderate the cadmium toxicity through the induction and accumulation of proteins related to detoxification of cadmium. These findings have broad implication for many cacao growing regions in South America and any region with toxic levels of cadmium in the soils.

3. Identification of positive correlations between soil, leaves and beans nutrient status and dry biomass of cacao beans and pod husk. Mineral nutrient management of soils and cacao is one of the main strategies used to increase the productivity in cacao production areas. Productivity, and mineral nutrient levels of cacao trees are directly related to nutrient status of the soil. Increased knowledge of mineral nutrient status of cacao can assist in designing an effective soil nutrient application strategy. ARS scientists from Beltsville, Maryland, with scientists from Cacao Research Institute (CEPLAC/CEPEC) Bahia, Brazil utilized an exploratory linear correlation analysis to predict relationships between the nutrient contents of the soil, leaves and beans of cacao trees with dry biomass of cacao beans and pod husks. High cacao productivity was positively correlated with the leaf concentrations of potassium, calcium, magnesium, iron, manganese, and zinc. This information will be useful to farmers, cacao researchers and extension workers to design efficient and economically acceptable soil nutrient management strategies to reduce plant mineral nutrient deficiencies and increase cacao bean yields in infertile acidic tropical soils.

Review Publications
Marrocos, P.C., Araujo, Q.R., Loureiro, G.A., Sodre, G.A., Ahnert, D., Baligar, V.C. 2020. Mineral nutrition of cacao (Theobroma cacao L.): Relationships between foliar concentrations of mineral nutrients and crop productivity. Journal of Plant Nutrition. 15 (11):2369–79.
Oliveira, B.R., Almeida, A.A., Pirovani, C.P., Barroso, J.P., Neo, C.H., Santos, N.A., Ahnert, D., Mangabeira, P.A., Baligar, V.C. 2020. Mitigation of Cd toxicity by Mn in young plants of cacao, evaluated by the proteomic profiles of laves and roots. Ecotoxicology and Environmental Safety. 29:340–358.
Santos, S.J., Almeida, A.F., Ahnert, D., Silva, N.M., Santo, M.L., Santos, N.A., Baligar, V.C. 2020. Foliar applied cuprous oxide fungicide induces physiological, biochemical and molecular changes in cacao leaves. Scientia Horticulturae. 265:109224.
Araujo, Q., Loureiro, G.A., Ahnert, D., Valdez, R., Baligar, V.C. 2020. Interactions between soil, leaves and beans nutrient status and dry biomass of beans and pod husk of forastero cacao: An exploratory study. Communications in Soil Science and Plant Analysis.
Song, L., Cao, B., Meinhardt, L.W., Bailey, B.A., Zhang, D. 2019. Genetic improvement of Chinese jujube for disease resistances: current status, knowledge gaps and research needs. Crop Breeding, Genetics and Genomics. 2019;1:e190015.
Patel, P., Zhang, D., Borthakur, D., Hazarika, M., Boruah, P., Barooah, R., Sabbapondit, S., Neog, N., Gogoi, R. 2019. Quality green tea (camellia sinensis l.) Clones marked through novel traits. Beverages. 5(4):63.
Zhang, D., Vega, F.E., Huawei, T., Johnson, E., Solano, W., Meinhardt, L.W. 2020. Accurate differentiation of green beans of Arabica and Robusta coffee using nanofluidic array of single nucleotide polymorphism (SNP) markers. Journal of AOAC International. 103:315–324.
Fister, A., Leamdro, M., Zhang, D., Marden, J., Tiffin, P., Depamphilis, C., Maximova, S., Guiltinan, M. 2019. Wide ranging differences in tolerance to Phytopthora palmivora in four genetic groups of cacao. Tree Genetics and Genomes. 16:1.