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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

2019 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Progress was made on all three objectives, under the NP301 except for one component of Sub-objective 1A (listed above). Under Sub-objective 1A progress was made in understanding spatial distribution of cacao genetic diversity in the Upper Amazon. In collaboration with U.S. Fine Chocolate Industry Association (FCIA), wild cacao population in the upstream region of Purus river in Peru was analyzed. The single nucleotide polymorphism (SNP) analytical result revealed a new germplasm group of cacao, which has not been represented in the international or national cacao collections. This result demonstrates that future collecting expeditions in this area will be needed to fill a gap of genetic diversity in current international cacao genebanks, as well as in national collections in Peru. For Sub-objective 1B, in collaboration with the Universidad Nacional Agraria de la Selva (UNAS) in Tingo Maria, Peru, a cacao collection of elite local farm selections with fine flavor and high productivity were characterized using SNP markers. Population membership and parentage were revealed for these farmer selections. High yielding clones with unique cocoa flavor profiles were identified. This research provided valuable insight into the genetic structure and quality attributes in indigenous farmer selections, targeting the international market needs in premium cocoa products. It facilitated in situ conservation of farmer selections having a distinct genetic profile and post harvesting qualities. Under Sub-objective 1C, progress was made in assessing the genetic integrity and parentage of the clonal series of cacao germplasm preserved in the International Cacao Collection at the Tropical Agricultural Research and Higher Education Center in Costa Rica (CATIE). These cacao clonal series (ARF, CC and PMCT) were developed in Costa Rica from 1928 to 1992, which account for approximately 20% of the cacao germplasm maintained in the CATIE genebank. Using SNP markers, the genetic identity of these clonal series were verified and their parentage were reconstructed based on their SNP profiles. The information generated in this study is essential to guide the effective conservation, exchange and use of the improved cacao clones in the CATIE International cacao collection. For Objective 2 progress was made under Sub-objective 2A, in the response of cacao genotypes from South America and Puerto Rico to abiotic stresses such as drought, low light and metal toxicity. Growth chamber experiments were conducted to evaluate the acid soil tolerant and intolerant cacao clones and at the ARS Tropical Agricultural Research Station, Puerto Rico, (TARS) cacao genotypes were evaluated for growth, physiology, and nutrient use efficiency responses to adequate and deficit soil moisture levels (drought). The nutrient use efficiency, drought tolerance indexes, growth and physiological parameters were determined. Plant potassium levels and their effects on cacao tolerance to soil water deficit was also accessed with scientists from State University of Santa Cruz (UESC), Bahia, Brazil. Metabolic functions of potassium in the plant are related to enzymatic activation and protein synthesis. Leaf proteins were quantified and some of these proteins were directly related to the mechanisms of drought tolerance, and were activated by the interactions between soil potassium levels and drought. There was a positive interaction observed between soil potassium, and protein synthesis, antioxidative metabolism, proline content and tolerance to drought in T. cacao plants. Under Sub-objective 2B progress was made in the evaluation of cacao germplasm for accumulation and translocation of cadmium and nutrient use efficiency in different cacao clones with scientists from the State University of Santa Cruz (UESC), and Cacao Research Institute (CEPLAC/CEPEC) Bahia, Brazil, Tropical Crop Research Institute (ICT) in Tarapoto, Peru and University of Florida Indian River Research and Education Center (IRREC) Fort Pierce, Florida through funded agreements. Collaborative research evaluated the mitigation of cadmium toxicity by zinc in young cacao plants. Plants physiological, biochemical, molecular and micromorphological responses were evaluated. An increase of Zinc concentration in the soil promoted the decrease of the cadmium uptake by the roots and reduced the transport of cadmium to the leaves. Further experiments were conducted under this sub-objective to verify the linear relationships between the foliar nutrient concentrations of 48 cropping areas. The cropping areas with very high productivity (equal to or higher than 1600 kg of dry cacao beans ha-1 year-1), were positively correlated with the leaf macronutrients (potassium, calcium, and magnesium), and the micronutrients (iron, manganese and zinc). Progress was made assessing abiotic stresses (drought, soil acidity, light quality and soil cadmium toxicity) of cacao varieties or genotypes collected from various Peruvian river basins, as well as national and international cacao genotypes. Various morphological, physiological plant traits have been recorded and the obtained data is being analyzed. Under Sub-objective 3, collaborative research on the molecular characterization of coffee germplasm was carried out in Côte d'Ivoire. Côte d'Ivoire is part of the center of origin and center of genetic diversity of many Coffea species, especially Coffea canephora (Robusta coffee). In 2019, a panel of SNP markers suitable for genotyping Robusta coffee was developed by ARS scientists based on a diverse set of coffee germplasm provided by Côte d'Ivoire National Agricultural Research Institute (CNRA), which maintains one of the largest coffee collections in the world (approximately 10,000 accessions). The SNP markers developed by this collaborative research are being applied to characterize Robusta coffee germplasm held in Côte d’Ivoire, Ghana and Robusta coffee germplasm maintained in the International Coffee Genebank maintained in Costa Rica. Additionally, progress was made in the development of a draft genome for dragon fruit (Hylocereus undatus). Dragon fruit is a cactus fruit species native to tropical America. This crop is highly water efficient and has a good adaptability to southern California, Florida, Hawaii and Puerto Rico. In collaboration with University of Nebraska Lincoln, USDA-ARS TARS and Dovetail Genomics, LLC, the first draft genome of dragon fruit was developed. The 1,332 Mbp genome of dragon fruit was sequenced using a combination of 10X Chromium sequencing and Chicago and Hi-C chromatin proximity ligation from Dovetail Genomics. It is estimated that over 87% of the dragon fruit genome was assembled into 33,691 scaffolds. A BUSCO (Benchmarking Universal Single-Copy Ortholog) analysis of the draft genome suggested that the draft genome has 88% of the single copy conserved genes. Additionally, progress was made under Objective 3 in developing SNP markers and improving genetic integrity in Chinese jujube (Ziziphus jujuba Mill). Chinese jujube is an economically important fruit tree with outstanding adaptability to marginal lands and a broad range of climate conditions. In the United states, jujube was reported as having good potential in regions such as the Southwest, where jujube’s late-spring bud break makes it a good alternative crop to fruits that suffer from late-spring frosts. In collaboration with Ningxia University, China, a total of 32,249 SNP markers were identified through the approach of data mining and a panel of 192 validated SNP markers were developed. These SNP markers provide a powerful tool for jujube germplasm management. Also under Objective 3 progress was made in accurate authentication of green beans of coffee. Arabica and Robusta coffee are the two main types of coffee traded in international market. In general, Arabica coffee is known to have better quality in terms of sensory characteristics; thus, it has a higher market value than Robusta coffee. Accurate differentiation of green beans of the two species is, therefore, of commercial interest in the coffee industry. Using the newly developed coffee SNP markers, we tested the efficacy of multi-locus SNP genotyping for authentication of green coffee beans. Unambiguous differentiation between Robusta and Arabica coffee was achieved using this approach, which is sufficiently robust to detect admixture of green coffee beans in a high-throughput fashion. Moreover, this approach can potentially identify varietal origin within Robusta and Arabica coffee. Finally, under the same objective we analyzed the genetic diversity in the Mississippi State University tea germplasm collection. Tea is a new specialty crop with great potential in Mississippi. Comparing with other alternative crops, tea is a better at resisting storm damage in this region. In collaboration with Mississippi State, the tea germplasm collection maintained at the University was analyzed using SNP markers developed by ARS scientists at Beltsville, Maryland. The results revealed the genetic background of the tea germplasm accessions, even though the introduced material lacked passport data. The SNP profiles verified genetic integrity in the seedling nursery and can be used to ensured true-to-type clonal propagation.

4. Accomplishments
1. Development of single nucleotide polymorphism (SNP) markers to support germplasm management of Chinese jujube (Ziziphus jujuba). Chinese jujube is an economically important fruit tree in Asia with outstanding adaptability to marginal lands and a broad range of climate conditions. In the United States, jujube was reported as having good potential in regions such as the Southwest, where jujube’s late-spring bud break makes it a good alternative crop to fruits that suffer from late-spring frosts. To utilize jujube in the U.S. as an alternative crop, superior germplasm would need to be used, however, a high rate of mislabeling in Chinese jujube germplasm restricts the sharing of information and materials among jujube researchers and hampers the use of jujube germplasm in breeding. In collaboration with Ningxia University, China, 5,800 SNP markers were developed through the data mining of genome sequences. After validation and testing, 192 reliable polymorphic SNP markers were identified and used to analyze 206 accession of jujube revealing high levels of mislabeling (40%) within the collection. These single nucleotide polymorphism (SNP) markers provide a powerful tool for accurate genotype identification and pedigree verification for jujube germplasm. This information will be useful to jujube growers and scientists to efficiently use jujube germplasm for cultivation and research and will advance the development of jujube as an alternative crop in the U.S. This technology is being used by scientists in New Mexico State University (NMSU) to fingerprint introduced jujube germplasm for improved genebank management and varietal selection, which will advance the development of jujube as an alternative crop in the U.S.

Review Publications
Santos, E.A., Almeida, A.F., Branco, M.C., Santos, I.C., Ahnert, D., Baligar, V.C., Valle, R.R. 2018. Path analysis of phenotypic traits in young cacao plants under drought conditions. PLoS One. 13(2):1-16.
Araujo, Q.R., Ahnert, D., Loureiro, G.A., Faria, J.C., Fernandes, C.A., Baligar, V.C. 2018. Soil quality index for cacao cropping systems. Archives of Agronomy and Soil Science. 64:1892-1909.
Nascimento, J.L., Almeida, A.F., Barroso, J.P., Mangabeira, P.O., Ahnert, D., Souza, A.G., Silva, J.V., Baligar, V.C. 2018. Physiological, ultrastructural, biochemical and molecular responses of young cocoa plants to the toxicity of cr (iii) in soil. Ecotoxicology and Environmental Safety. 159:272-283.
Baligar, V.C., Elson, M.K., He, Z.L., Li, Y.C., Paiva, A.D., Ahnert, D. 2018. Growth, physiological and nutrient uptake traits of Crotalaria cover crops influenced by levels of carbon dioxide under low light Intensities. International Journal of Plant Sciences. 23(1):1-14.
Araujo, Q.R., Loureiro, G.A., Rohsius, C., Baligar, V.C. 2018. Impact of soils and cropping systems on biochemical attributes of dry cacao beans. Agrotropica: Revista de Agricultura dos Tropicos Umidos. 30(3):175-194.
Ahnert, D., Melohellen, L., Santos Francisca, F.J., Lima, L., Baligar, V.C. 2018. Breeding and cacao productivity in Brazil (in portuguese) (melhoramento genético e produtividade do cacaueiro no brasil). In: Souze Junior, J.O., editor. Cocoa: cultivation, research and innovation. Ilheus, Bahia, Brazil: Editus. p.151-181.
Mata, A., Arciniegas, A.L., Phillips, W.M., Meinhardt, L.W., Mischke, B.S., Zhang, D. 2018. Assessing hidden parentage and genetic integrity of the “United Fruit Clones” of cacao (Theobroma cacao) from Costa Rica using SNP markers. Breeding Science. 68:545-553.
Laliberte, B., End, M., Cryer, N., Daymond, A., Engels, J., Esks, A., Gilmour, M., Lachenaud, P., Phillips-Mora, W., Turnbull, C., Umaharan, P., Zhang, D., Weise, S. 2018. Conserving and exploiting cocoa genetic resources: the key challenges. In: Umaharan, P., Achieving sustainable cultivation of cocoa, Cambridge, UK: Burleigh Dodds Science Publishing. 25 p.
Arevalo, E., Zhou, L., Meinhardt, L.W., Motilal, L., Mischke, B.S., Zhang, D. 2019. Genetic identity and origin of ‘Piura Porcelana’ – a fine-flavored traditional variety of cacao (Theobroma cacao) from the Peruvian Amazon. Tree Genetics and Genomes. 15:11.
Paiva, A.Q., Arevalo-Hernandez, C.O., Baligar, V.C., Fernandes, R.B. 2018. Atributos físicos do solo para a cultura do cacaueiro [Physical attributes of soil for the culture of cacao] (In Portuguese). In: Souza Junior, J.O. Cocoa: Cultivation, Research and Innovation, Ilheus, Bahia, Brazil: Editus. p. 215-233.