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

2021 Annual Report

Objectives
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.

Approach
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
Considerable progress was made under Sub-Objective 1A: In collaboration with scientists from the Colombian Agricultural Research Corporation, a total of 5280 trees, representing the entire Colombian national cacao collections maintained in Palmira, as well as the backup collection in La Suiza, Colombia, were genotyped using a genetic marker panel developed by ARS scientists. The analysis led to the identification of 132 synonymous groups, including 460 individual trees, in the Colombian Cacao Genebank. The result also revealed a total of 186 plots that have mixed genotypes, showing the mislabeling in the genebank. Based on the data, the true-to-type identity of the introduced international clones was also verified. These results led to the correction of mislabeled cacao clones, significantly improving the integrity of the collection. Moreover, the results provide the scientific guidance to reduce genetic redundancy in this collection and improve future genetic diversity analysis in Colombia. Progress was made under Sub-Objective 1B: In collaboration with scientists from Yunnan Forestry College, China, ARS scientists analyzed a cacao collection maintained in the Xishuangbanna Tropical Botanical Garden in Yunnan, China, with single nucleotide polymorphic (SNP) markers. The study demonstrated that the cacao genetic resources in Yunnan have a genetic background mainly composed of Amelonado and Trinitario and lack multiple sources of resistance to major cacao diseases. The present results suggest that new introductions are needed to enrich genetic diversity in this collection and improve cacao production in this tropical region of China. Under Sub-Objective 1C, progress was made in developing a population informative genotyping panel for cacao. In collaboration with ARS scientists in Sub-Tropical Horticulture Research Station in Miami, ARS scientists in Beltsville, Maryland, validated a new genotyping panel for cacao germplasm characterization. This new SNP panel is more ancestry-informative than the previous one, thus providing improved accuracy for revealing the origin of each cacao accession. Scientists are now applying the new panel to genotype the ARS cacao repository maintained in Mayaguez, Puerto Rico, and the International Cocoa Quarantine Centre located in Reading, United Kingdom. SNP marker profiles generated by this genotyping panel will serve as designated reference genotypes for the international cacao community. Due to imposed restrictions by local and national authorities due to the COVID-19 epidemic, progress was limited with collaborating scientists from the State University of Santa Cruz, Bahia, Brazil, Tropical Crop Research Instituto Tarapoto, Peru, IRREC- UFL Fort Pierce, Forida, and Colombian Corporation for Agricultural Research at Center for Investigación, Tibaitatá, and Palmira. Progress was made on Sub-objective 2A. Completed data analysis of the following research experiments: (a) Responses of six perennial legume cover crops to ambient and elevated levels of carbon dioxide (CO2) under low levels of photosynthetic photon flux density (PPFD) were evaluated for growth, physiological changes, and nutrient uptake (b) Seven genetically unique cacao genotypes were evaluated for their response to ambient and elevated levels of CO2 under inadequate to adequate levels of PPFD and (3) experiments were done to assess the responses of 4 cacao genotypes to soil water deficit. Progress was made under Sub-objective 2B. (a) ARS scientists analyzed the responses of cacao genotypes from Puerto Rico to low and to toxic levels of soil Cadmium (Cd). With collaborating scientists from the State University of Santa Cruz, Bahia, Brazil, Tropical Crop Research Instituto in Tarapoto, Peru, Indian River Research and Education Center- University of Florida, Fort Pierce, Florida, and Corporation for Agricultural Research at Tibaitatá and Palmira in Colombia the following experiments were conducted: (a) Greenhouse studies in Peru with 58 cacao genotypes selected from 4 geographically diverse groups were conducted to assess the effects of 80% shade and 50% shade on growth, physiological, nutrient uptake efficiency. (b) In Brazil, bean samples from the PH-16 cacao genotypes were sampled from 12 cacao agroforestry systems to evaluate the impact of growing conditions on the amount and types of free amino acid in the beans. (c) In Florida, sorption and incubation experiments were conducted to compare the effectiveness of dolomite phosphate rock, humic acid-activated dolomite phosphate rock, and biochar on the immobilization of cadmium and lead in soils. The amounts of Cd and lead (Pb) were negatively correlated with soil pH and available phosphorus, indicating that the amendments were the dominant mechanism for enhanced immobilization of metals in the contaminated soils. (d) In Colombia, the following experiments are in progress: (i) Soil incubation experiment to determine the effectiveness of inorganic and organic soil amendments for reducing plant-available Cd is being conducted. (ii) Field experiments are in progress to determine the total and bioavailable soil Cd and their relationship to soil properties and cacao leaf and bean Cd contents from various cacao production regions of Colombia. (iii) Research is underway in Colombia to study the effects of the rootstock on cadmium uptake by cacao scion. Significant progress was made under Objective 3. Studies were carried out in collaboration with scientists from the World Coffee Research to investigate the genetic variation within and among Arabica coffee varieties. Coffee leaf samples were collected from nurseries across seven countries in Latin America, including El Salvador, Guatemala, Honduras, Panama, Costa Rica, Puerto Rico, and Peru. Molecular marker profiles for these samples were generated using an ARS-developed DNA fingerprinting panel. Unique varietal profiles were detected for all tested varieties, demonstrating the effectiveness of the technology to differentiate coffee varieties. The observed difference among varieties was sufficiently large to enable the detection of mislabeled trees within each variety. Most of the tested coffee varieties are not genetically fixed pure lines. On average, 10% of the SNP markers were heterozygous. This variety difference suggested the varying extent of fixation in different varieties of Arabica coffee. The within-variety difference is small in old varieties such as “Typica” and “Bourbon”. A larger difference was observed in the introgressed varieties. The study demonstrated that using the new genetic tool developed by ARS, “true-to-type” varietal profiles can be established for Arabica coffee, making it feasible to obtain “pure lines” of Arabica through selection and seed propagation. The results also have broad downstream applications, such as improving the accuracy and efficiency in genebank management, supports nursery and seed garden verification, and assesses authenticity and traceability of coffee beans and coffee products in the value chain. With scientists from the Cocoa Research Institute of Ghana, ARS scientists analyzed how information on genetic diversity can be used to select promising parental clones for coffee breeding. The study was based on field trials of 62 bi-parental families over 10 years. SNP markers were used to measure genetic distances among parental clones. A highly significant positive correlation was observed between genetic distances between parents and the cumulated yield performance of hybrid families. The results suggest that genetic distances measured by SNP markers can be used to select superior parental combinations for hybrid progenies. The efficiency of the Robusta coffee breeding program can therefore be improved by exploring heterosis between two divergent parental clones. Progress was made in molecular characterization of coffee germplasm collection in Hawaii with scientists from Hawaii Agriculture Research Center and ARS Tropical Plant Genetic Resources and Disease Research Lab in Hilo, Hawaii. The coffee collection maintained in Hilo, Hawaii, was characterized using SNP markers developed by ARS scientists. The result clarified the genetic identity of the introduced coffee germplasm and parentage of improved varieties in Hawaii. The results also confirmed the genetic identity of Kona coffee, a world-renowned coffee that is exclusively grown in Hawaii. The information is highly useful for establishing a coffee germplasm collection in Hawaii and provides scientific data to support the coffee industry in Hawaii. Progress was made in improving genebank management of Chinese jujube (or red date). In collaboration with a scientist in the Alcalde Agricultural Science Center, New Mexico State University, their entire collection of Chinese jujube genetic resources was genotyped using ARS-developed SNP markers. The result led to the detection of mislabeled accessions and reduced genetic redundancy in this collection. Jujube is a popular tree fruit crop for arid and semiarid areas of the temperate and subtropical regions where other common fruit trees do not grow well. Jujube is cultivated in more than 20 states in the U.S. Progress was made in developing genomic resources for dragon fruit, a popular tropical fruit in the cactus family. In collaboration with the University of Nebraska, Lincoln, and ARS tropical agricultural research station in Mayaguez, ARS scientists in Beltsville, Maryland, published the first draft genome of dragon fruit. This tropical cactus crop is resistant to drought and contains high amounts of antioxidants, vitamins, and minerals. Gene clusters for drought resistance and fruit flavor were found in this chromosomal level draft genome, which was used to identify 3,000 high-quality SNP markers, that will be used for molecular characterization of dragon fruit genetic resources.

Accomplishments
1. Development of a chromosomal lengthen draft genome of dragon fruits. Dragon fruits (Pitaya, Pitahaya) are important for agricultural industries in many countries, particularly in Asia and Latin America, and as a new alternative crop in the USA. In collaboration with researchers from the University of Nebraska, Lincoln, and ARS scientists in Mayaguez, Puerto Rico, ARS scientists in Beltsville, Maryland, published the first chromosomal lengthen draft genome of dragon fruit (Hylocereus undatus). This tropical cactus crop is resistant to drought and contains high amounts of antioxidants, vitamins, and minerals. Gene clusters for drought resistance and fruit flavor were found in this chromosomal level draft genome. The genome also enabled the identification of 3,000 high-quality SNP markers, which will be used for molecular characterization of dragon fruit genetic resources. This information will be used by researchers, plant breeders, and collection curators worldwide to study and improve thisdrought-resistant tropical cactus fruit crop and advance its utilization and production.

2. Molecular characterization of Chinese Jujube germplasm collection in New Mexico. Chinese jujube (or red date) is a popular tree fruit crop for arid and semiarid areas of temperate and subtropical regions where other common fruit trees do not grow well. Jujube is highly adaptable to adverse growing conditions and is cultivated in more than 20 states in the U.S., with the leading producers being New Mexico and Texas. In collaboration with a scientist at New Mexico State University, ARS scientists in Beltsville, Maryland, analyzed the entire collection of jujube genetic resources maintained at New Mexico State University using genetic markers developed by ARS. The results lead to the detection of mislabeled accessions and reduced genetic redundancy in this collection. Jujube researchers in the USA have used this information to improve germplasm management for this alternative fruit crop for American producers.

3. Molecular characterization of coffee germplasm collection in Hawaii. Hawaii and Puerto Rico are the only regions in the USA where commercial coffee production occurs, and in both regions, the coffee is produced for the premium market. To improve coffee production and selection of premium coffee USDA is developing coffee germplasm collections. In collaboration with scientists from Hawaii Agriculture Research Center and an ARS scientist from Hilo, Hawaii, ARS scientists in Beltsville, Maryland, characterized the coffee collection maintained in Hilo, Hawaii, using genetic markers developed by ARS scientists. The results clarified the genetic identity of the introduced coffee germplasm and parentage of improved varieties in Hawaii. The results also confirmed the genetic identity of Kona coffee, a world-renowned coffee that is exclusively grown in Hawaii. The information is beneficial for expanding the establishment of a coffee germplasm collection in Hawaii. It also provides scientific data to support the coffee industry in Hawaii, where coffee production is an important part of the local economy.

4. Identification of low cadmium accumulating cacao genotypes. Soils in the cacao growing region of South America typically contain high levels of cadmium, and uptake of cadmium by cacao beans represents a risk for cacao exportation and to consumers affecting the economic well-being of the resource-poor-cacao producers in South America. ARS scientists in Beltsville, Maryland, with scientists from the Tropical Crop Research institute in Tarapoto, Peru, identified low cadmium accumulating wild and domesticated cacao genotypes. Low accumulating genotypes contained approximately 30% less cadmium than control genotypes. Such information will be helpful to plant breeders to breed less cadmium, accumulating cacao cultivars, thereby reduce cadmium in cacao beans. Farmers can use these low cadmium accumulating cultivars in the production area where cadmium toxicity is an issue.

5. Humic acid activated dolomite phosphate rock remediates toxic levels of cadmium and lead in soils. Soils polluted by toxic metals such as cadmium and lead are a severe environmental and human health issues. ARS scientists from Beltsville, Maryland, with scientists at the University of Florida in Fort Pierce, Florida, found that humic acid-activated dolomite phosphate rock has the potential for remediating toxic levels of cadmium and lead in contaminated soils. The addition of this compound to soils reduced the available levels of these heavy metals to the plants, with cadmium reduced by 87% and lead decreased by 76 percent. This information will be helpful to researchers and soil remediation workers to develop technology to limit the uptake of toxic metals in contaminated soils by plants, thereby reducing the entry of these toxic metals into the food chain.

Review Publications
Ma, Y., Song, L., Wang, Z., Zhang, D., Cao, B. 2021. Fruit morphology measurements of Jujube Cultivar ‘Lingwu Changzao’ (Ziziphus jujuba Mill. cv. Lingwuchangzao) during fruit development. Horticulturae. https://doi.org/10.3390/horticulturae7020026.
Gardini, G.E., Arevalo-Hernandez, C.C., Barraza, F., Farfan, A., He, Z.L., Baligar, V.C. 2020. Growth and nutritional responses of wild and domesticated cacao genotypes to soil Cd stress. Science of the Total Environment. https://doi.org/10.1016/j.scitotenv.2020.144021.
Baligar, V.C., Elson, M.K., He, Z., Li, Y., Paiva, A.D., Ahnert, D., Almeida, A.F. 2020. Light intensity effects on growth, physiological and nutritional parameters of tropical perennial legume cover crops. Agronomy. https://doi.org/10.3390/agronomy10101515.
Muñoz, J.A., Almeida, A.F., Pirovani, C.P., Ahnert, D., Baligar, V.C. 2021. Mitigation of Pb toxicity by Mn in seedling of the cacao clonal CCN 51 genotype grown in soil: physiological, biochemical, nutritional and molecular responses. Ecotoxicology. https://doi.org/10.1007/s10646-021-02348-y.
Zheng, J., Meinhardt, L.W., Goenaga, R.J., Zhang, D., Yin, Y. 2021. The chromosome-level genome of dragon fruit reveals whole-genome duplication and chromosomal co-localization of betacyanin iosynthetic genes. Horticulture Research. https://doi.org/10.1038/s41438-021-00501-6.
Jiang, W., Li, N., Zhang, D., Meinhardt, L.W., Cao, B., Li, Y., Song, L. 2020. Elevated temperature and drought stress significantly affect fruit quality and activity of anthocyanin-related enzymes in jujube (Ziziphus jujuba mill. cv. ‘Lingwuchangzao’). PLoS ONE. https://doi.org/10.1371/journal.pone.0241491.t003.
Akpertey, A., Padi, F., Meinhardt, L.W., Zhang, D. 2021. Effectiveness of Single Nucleotide Polymorphism Markers in Genotyping Germplasm Collections of Coffea canephora Using KASP Assay. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2020.612593.
Zhang, D., Vega, F.E., Solano, W., Fuyuan, S., Infante, F., Meinhardt, L.W. 2021. Selecting a core set of nuclear SNP markers for molecular characterization of Arabica coffee (Coffea arabica L.) genetic resources. Conservation Genetics Resources. https://doi.org/10.1007/s12686-021-01201-y.
Baligar, V.C., Elson, M.K., He, Z., Li, Y., Paiva, A.D., Almeida, A.A., Ahnert, D. 2021. Impact of ambient and elevated [co2] in low light levels on growth, physiology and nutrient uptake of tropical perennial legume cover crops. Plants. https://doi.org/10.3390/plants10020193.
Baligar, V.C., Elson, M.K., Almeida, A., Araujo, Q.R., Ahnert, D., He, Z. 2021. The impact of ambient and elevated [CO2] and low to adequate photosynthetic photon flux density on growth, physiology and nutrient use efficiency of juvenile cacao genotypes. Agronomy. https://doi.org/10.3390/agronomy11020397.
Liu, B., He, Z., Liu, R., Montenegro, A.C., Ellis, M., Li, Q., Baligar, V.C. 2020. Comparative effectiveness of activated dolomite phosphate rock and biochar for immobilizing cadmium and lead in soils. Chemosphere. https://doi.org/10.1016/j.chemosphere.2020.129202.
Santos, M.L., Almeida, A., Silva, N.M., Silva, J.V., Barroso, J.P., Souza, J.O., Ahnert, D., Baligar, V.C. 2020. Mitigation of cadmium toxicity by zinc in young cacao plants. Environmental and Experimental Botany. https://doi.org/10.1016/j.envexpbot.2020.104201.
Marrocos, P.C., Loureiro, G.A., Araujo, Q.R., Sodre, G.A., Ahnert, D., Baligar, V.C. 2020. Mineral nutrient ratios and cacao productivity. Journal of Plant Nutrition. https://doi.org/10.1080/01904167.2020.1771582.
Arevalo-Gardini, E., Canto, J., Alegre, J., Loli, O., Julca, A., Baligar, V.C. 2020. Cacao agroforestry management systems effects on soil fungi diversity in the Peruvian Amazon. Ecological Indicators. https://doi.org/10.1016/j.ecolind.2020.106404.