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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

2022 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
Under the Sub-Objective 1A, significant progress was made in understanding cacao genetic diversity and evolution through comparative genomic analysis. ARS scientists, in collaboration with scientists in the University of Nebraska-Lincoln, developed three chromosome-level genomes of cacao cultivars (IMC 7, Scavina 6 and Pound 7), which came from three important populations from Upper Amazon – the center of origin of cacao. Gene prediction revealed 20,942, 21,001, and 20,623 protein-coding genes and the BUSCO scores of 94.4%, 95.1% and 89.2% for Pound 7, IMC 67 and SCA 6, respectively. Chromosome synteny comparisons detected larger structural variations (duplication, translocation, and inversion) in Chr3, Chr6, Chr7, Chr8, Chr10 between the three new genomes and the previously published Criollo genome, and in Chr7, Chr8, Chr9, Chr10 between the three new genomes and the published Matina genome. In total, 2,757,719 high confident single nucleotide polymorphisms (SNPs) and insertion-deletion mutations (INDELs) were identified and supported by all three new genomes. Result of gene ontology enrichment analysis revealed key genes that have undergone the most significant sequence divergence due to environmental adaptation. Significant progress was made under Sub-Objective 1B: In collaboration with scientists from the Tropical Crop Institute (ICT), Peru and Cacao Research Center (CRC), University of West Indies, Trinidad, 3600 cacao trees were genotyped using targeted Genotyping by Sequencing with 450 SNP markers. The analyzed samples include all known wild cacao populations, farmer varieties and landraces in tropical America. The analytical result confirmed the previous observation regarding the new genetic groups and sub-groups in the primary gene pool of T. cacao, which complementing the existing national and international cacao collections. Under the same Sub-Objective, progress was made in collaboration with scientists from the University of the South Pacific, Alafua Campus, Samoa, and the Department of Agriculture and Fisheries, Queensland, Australia. A cacao collection comprising elite farmer selections from Makira Island, Solomon Islands, was analyzed using SNP markers. The result, based on both genetics and morphological traits, revealed that Amelonado is the dominant type of cacao cultivated in Makira Island. Moreover, the significant phenotypic variation within the Amelonado accessions suggests that there were multi-source introductions of Amelonado into this region. Today, cocoa is an important export earner and source of rural livelihoods for the Makira Islanders. This result provides a scientific baseline information for sustainable preservation of cacao germplasm and production of fine flavored cocoa in the region of South Pacific. Under Sub-Objective 1C, progress was made in verification of parental clones of seeds gardens in Ivory Coast. In collaboration with scientists in National Agricultural Research Center (CNRA), Ivory Coast, a total of 2096 cacao trees genotyped using SNP markers were analyzed to ascertain genetic integrity of the released cocoa hybrids. Off type were found in all the seed gardens analyzed with a predominance of mis-identification of genotypes. The genetic purity of seeds produced varied from 82% to 100%. The genotyping data, as well as the analytical results were submitted to the International Cacao Germplasm Database (ICGD), Reading, U.K. In collaboration scientists from AGROSAVIA, Colombia, progress was made in submitting the genotyping data of the 5280 trees, representing the entire collection, to the International Cacao Germplasm Database (ICGD). Sub-Objective 2. 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, (UESC) Bahia, Brazil, Tropical Crop Research Instituto (ICT) Tarapoto, Peru, IRREC- UFL Fort Pierce, Florida, and Colombian Corporation for Agricultural Research at Center for Investigación (AGROSAVIA),Tibaitatá, and Palmira, Colombia. Substantial progress was made to address Sub-Objective 2A. At USDA Beltsville, three genetically contrasting cacao genotypes were evaluated for their response to different carbon dioxide concentrations (400 to 700 µmol·mol-1) and light intensities (PPFD from 100 to 400 µmol·m-2·s-1). Significant difference was detected in growth and nutrient use efficiency at ambient and elevated levels of carbon dioxide under low to adequate levels of light intensities. The result showed that elevated CO2 and adequate light intensity are beneficial in improving cacao growth and mineral nutrient uptake and use efficiency. At the Institute of Tropical Crops (ICT) Peru, greenhouse study was undertaken to evaluate cacao genotypes for physiological, nutrient-uptake-efficiency and shade-tolerance under different light intensities (50% and 80% shade where photosynthetic photon flux density (PPFD) was 1000 and 400 µmol). Significant genotype difference was observed for growth, physiological and nutritional traits, and tolerance to shade. Ten genotypes with superior tolerance to shade were selected. At the State University of Santa Cruz (UESC), Bahia, Brazil, field experiment was conducted to evaluate different edaphic crop conditions on the free amino acid profile of dry beans of the “PH-16” cacao clone. The hydrophobic free amino acids were found the largest contributor of the observed variation, followed by Glutamic acid as the second (13.09%), in terms of the free amino acid profiles. The result showed that cacao genotype is the primary source of variation, whereas the soil conditions had minimum impact on free amino acids contents of cacao beans. This information will be useful for cacao producer to consider the genotype as the primary source of variation in the quality of cacao beans and ultimately the chocolate to be produced. Substantial progress was made under Sub-Objective 2B. In collaboration with scientists in the Tropical Crop Research institute (ICT) in Tarapoto, Peru, greenhouse experiment was conducted to evaluate 53 cacao genotypes for their response to soil Cd stress. Significant Cd tolerance was observed among cacao genotypes and seven promising clones with low cadmium accumulation were selected. The identified clones contained as low as 8.8% of the Cd concentration relative to the high Cd clones. These low Cd clones thus can be used as parental lines for breeding low Cd varieties or as root stock. In collaboration with scientists in AGROSAVIA, Colombia, significant root-stock effect on cadmium and nutrient uptake was detected in juvenile cacao, based on the evaluation result of 320 progenies. Moreover, there was a significant interaction of rootstock × scion × soil, in terms of Cd and nutrient uptake, early growth, and photosynthetic process in juvenile cacao plants. 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. This information will be useful to plant breeders to breed less cadmium accumulating cacao cultivars for Latin American cocoa producers. Progress was made under Objective 3. In collaboration with scientists in ARS Tropical Agriculture Research Station (TARS), Mayaguez and the World Coffee Research (WCR), ARS scientist analyzed coffee germplasm collections and nurseries in Puerto Rico. The result led to the identification of two most important Puerto Rican coffee varieties - Limani and Fronton. The result contributed to the recovery of the island’s coffee production, most of which was destroyed due to the damage of Hurricane Maria in 2017. In collaboration with scientists in the Center for Tropical Agricultural Research and Education (CATIE), Costa Rica and World Coffee Research, ARS scientists genotyped 1820 coffee trees from the international coffee genebank using DNA markers. The result led to the identification of redundant accessions in the international collection and enabled the selection of a core collection, which will facilitate the establishment of a bank-up collection for the International Coffee genebank. In collaboration with scientists from National Agriculture Research Institute, Ivory Coast and the French Agricultural Research Centre for International Development, DNA markers were used to assess pollen contamination rate in the progenies of a native Guinean population of Robusta coffee in Ivory Coast. The results show that the introduced Congolese coffee trees naturally hybridized with the endemic Guinean Robusta coffee trees in Ivory Coast. The mismatched flowering time in these two genetic groups is not a sufficient barrier to genetic crossing. While these inter-group hybrids positively contribute to coffee production in Ivory Coast, they also cause genetic erosion in the wild Guinean gene pool. The result showed that an integrative approach needs to be developed to alleviate this threat. This study provides baseline information for coffee researchers, breeders and genebank curators, to study and improve the conservation of Robusta coffee genetic resources. Significant progress was made in developing new genomic resources for other tropical perennial crops. In collaboration with scientists from the University of Nebraska-Lincoln, the draft genome of Cherimoya (Annona cherimola), an important fruit species of the Annonaceae family, was developed. The genome assembly has a size of 794 Mb and consists of 1,377 scaffolds with a scaffold N50 = 97.59 Mb. A total of 45,702 protein-coding genes are predicted with 93.7% of complete BUSCOs as the gene content completeness. The cherimoya genome, the second sequenced genome of the Annonaceae family, will be a valuable resource to study the genetic diversity and evolution of flowering plants.

1. The development of draft genomes for three cacao varieties from Upper Amazon. In collaboration with scientists in the University of Nebraska-Lincoln, ARS scientists in Beltsville, Maryland, developed three new chromosome-level genomes of cacao cultivars (IMC 7, Scavina 6 and Pound 7). These varieties represent three important populations from the Upper Amazon – the center of origin of cacao. No draft genome of Upper Amazon wild cacao is available, and these high-quality genomes will serve as important genomics resource for discovery of genetic markers and genes conferring important phenotypic traits, such as disease resistances and quality attributes. This information will be used by cacao researchers, plant breeders, and collection curators, around the world, to improve germplasm management and by cacao plant breeding improvement programs.

2. Development of a new genome for Annona cherimola. Genomic resources for economically important tropical fruit crops are lacking in many species. In collaboration with scientists from the University of Nebraska-Lincoln, ARS scientists in Beltsville, Maryland, developed the first draft genome of Cherimoya (Annona cherimola). The genome assembly has a size of 794 Mb and a total of 45,702 genes were identified. The cherimoya fruit, also known as custard apple, is becoming increasingly popular in the U.S. market and the fruit is now produced in Puerto Rico and California. This draft genome will be a highly valuable resource for molecular marker development, genetic diversity analysis and genetic mapping for Cherimoya, as well as for other species in the Annonaceae plant family. This information will be used by researchers, plant breeders, and collection curators, around the world, to study and improve this fruit crop and advance its utilization and production.

Review Publications
Zheng, J., Meinhardt, L.W., Goenaga, R.J., Matsumoto Brower, T.K., Zhang, D., Yin, Y. 2022. The chromosome-level rambutan genome reveals a significant role of segmental duplication in the expansion of resistance genes. Horticulture Research. 9:uhac014.
Pirro, S., White, D., Bailey, B.A., Meinhardt, L.W. 2022. Genome assemblies of Erythroxylum. coca var. ipadu and Erythroxylum novogranatense var. truxillense. F1000Research. 11:457.
Baligar, V.C., Elson, M.K., Almeida, A., Araujo, D., Ahnert, D., He, Z. 2021. Carbon dioxide concentration and light levels on growth and mineral nutrition of juvenile cacao genotypes. American Journal of Plant Sciences. 12:818-839.
Arevalo-Gardini, E., Farfan, A., Barraza, F., Arevalo-Hernandez, C.0., Alegre, J., Baligar, V.C. 2021. Growth, physiological, nutrient uptake efficiency and shade tolerance responses of cacao genotypes under different shades. Agronomy. 11:1536.
Araujo, Q.R., Loureiro, G.A., Povoas, C., Steinmacher, S., Santos, S.S., Ahnert, D., Baligar, V.C. 2021. Effect of different edaphic crop conditions on the free amino acid profile of the PH-16 dry cacao beans. Agronomy. 11(8):1637.
Gnapi, E.D., Pokou, D., Legnate, H., Zhang, D., Montagnon, C., Bertrand, B., N'Guetta, S. 2022. Is the genetic integrity of wild coffea canephora from ivory coast threatened by hybridization with introduced coffee trees from central africa? Euphytica. 218:62.
Akpertey, A., Padi, F., Meinhardt, L.W., Zhang, D. 2022. Relationship between genetic distance based on single nucleotide polymorphism markers and hybrid performance in C. canephora. Plant Breeding.
Song, L., Zhang, H., Li, Z., Meinhardt, L.W., Zhang, D. 2021. Assessing genetic integrity of Chinese jujube cultivars using Single Nucleotide Polymorphism (SNP) markers. Agronomy. 11:2303.
Galvez, L.C., Koh, R.B., Barbosa, C.C., Asunto, J.C., Catalla, J.L., Atienza, R.G., Costalesa, K.T., Aquino, V.M., Zhang, D. 2021. Sequencing and de Novo Assembly of Abaca (Musa textilis Née) var. Abuab Genome. Genes. 12:1202.