Cacao Germplasm Research Articles

Genetic Diversity and Population Structure of Cacao (Theobroma cacao L.) Germplasm from Sierra Leone and Togo Based on KASP–SNP Genotyping

Cacao (Theobroma cacao L.) is a tropical tree species belonging to the Malvaceae, which originated in the lowland rainforests of the Amazon. It is a major agricultural commodity, which contributes towards the Gross Domestic Product of West African countries, where it accounts for about 70% of the world’s production. Understanding the genetic diversity of genetic resources in a country, especially for an introduced crop such as cacao, is crucial to their management and effective utilization. However, very little is known about the genetic structure of the cacao germplasm from Sierra Leone and Togo based on molecular information. We assembled cacao germplasm accessions (235 from Sierra Leone and 141 from Togo) from different seed gardens and farmers’ fields across the cacao-producing states/regions of these countries for genetic diversity and population structure studies based on single nucleotide polymorphism (SNP) markers using 20 highly informative and reproducible KASP–SNPs markers. Genetic diversity among these accessions was assessed with three complementary clustering methods, including model-based population structure, discriminant analysis of principal components (DAPC), and phylogenetic trees. STRUCTURE and DAPC exhibited some consistency in the allocation of accessions into subpopulations or groups, although some discrepancies in their groupings were noted. Hierarchical clustering analysis grouped all the individuals into two major groups, as well as several sub-clusters. We also conducted a network analysis to elucidate genetic relationships among cacao accessions from Sierra Leone and Togo. Analysis of molecular variance (AMOVA) revealed high genetic diversity (86%) within accessions. A high rate of mislabeling/duplicate genotype names was revealed in both countries, which may be attributed to errors from the sources of introduction, labeling errors, and lost labels. This preliminary study demonstrates the use of KASP–SNPs for fingerprinting that can help identify duplicate/mislabeled accessions and provide strong evidence for improving accuracy and efficiency in cacao germplasm management as well as the distribution of correct materials to farmers.

First Report of Cacao Mild Mosaic Virus Associated with Cacao (Theobroma cacao) in Hawai'i, USA.

Cacao, Theobroma cacao, is an important tropical agricultural crop and the key ingredient of chocolate, which has an international trade value of $103 billion/year (Marelli et al. 2019). Cacao mild mosaic virus (CaMMV) is a badnavirus that causes mild symptoms compared with some of the closely related species of Cacao swollen shoot virus (CSSV), the latter of which are currently only found in West Africa (Marelli et al. 2019; Ullah et al. 2021). CaMMV was recently found in symptomatic commercial cacao trees in Mayagüez, Puerto Rico (Puig et al. 2020), and subsequently detected in a USDA ARS (Agricultural Research Service) quarantine greenhouse in Miami, FL (Puig 2021). The USDA ARS germplasm repository in Hilo, Hawai'i, USA serves as a backup collection for Puerto Rico's cacao germplasm, and field trials have been established from cacao germplasm from Miami to evaluate select varieties. To determine if CaMMV is present in the collection in Hilo, greenhouse and field accessions were tested. Using an optimized sampling and PCR protocol established by Puig (2021), three young cacao petioles per tree or seedling were collected and pooled, and DNA was extracted using the NucleoSpin Plant II commercial kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Samples were molecularly identified via end point PCR, gel electrophoresis, and Sanger sequencing. PCR amplification of CaMMV using the virus-specific primer set Mia-1396F (5'-ACCGTGTCTAYCAGCACTGGA-3') and Mia-1667R (5'-GACCACCGTCAGCCAGAC-3') produced 289 bp amplicons. Of 230 plants sampled, 26 CaMMV positive detections were discovered in greenhouse and field plantings in Hilo. Most CaMMV+ plants contained some form of leaf chlorosis (96.2%). The sequenced PCR products from Hawai'i were deposited in GenBank (accession nos. OQ692890-OQ692891) and showed 99.2% nucleotide identity to CaMMV accessions from Puerto Rico (MW052520; n = 23) and 98.1% nucleotide identity to CaMMV accessions from Florida, USA (MZ409692; n = 3) in BLASTn analysis. For species-level confirmation, the RT-RNase H domain was amplified from 9 isolates using CaMMV-specific primers (Mia5385F, 5'-AGGACAACGGCTTTCTTGGT-3'/Mia6616R, 5'-GAGACTAACTTGGTTAGGGCT-3'), sequenced, and deposited in GenBank (accession nos. PP997461-PP997462). Sequences matched most closely to GenBank isolates from Puerto Rico (MT253659; 98.0%; n = 7) and Trinidad and Tobago (NC_033738; 97.1%; n = 2). CaMMV, previously known as cacao Trinidad virus A, was first reported in 1943 and was associated with 7 to 33% yield reduction, loss of vigor, and tree decline (Posnette 1944; Swarbrick 1961). CaMMV is known to exist in the Americas and was most recently detected in Brazil and Indonesia (Kandito et al. 2022; Ramos‑Sobrinho et al. 2021). To the best of our knowledge, this is the first report of CaMMV infecting cacao in Hawai'i. The cacao industry continues to expand in Hawai'i, and cultivation occurs on at least four of the main islands including Hawai'i Island, O'ahu, Maui, and Kaua'i. To develop disease management strategies, further investigation is needed to define CaMMV symptomology, and determine the distribution and effect this virus has on production in Hawai'i. In the meantime, cacao will be screened regardless of visible symptomology to maintain pathogen-free accessions and avoid the transfer of virus-containing germplasm.

Molecular Surveillance, Prevalence, and Distribution of Cacao Infecting Badnavirus Species in Côte d'Ivoire and Ghana.

The cacao swollen shoot disease (CSSD) caused by a complex of badnavirus species presents a major challenge for cacao production in West Africa, especially Ghana and Côte d'Ivoire. In this study, CSSD species detection efficiency, diversity, and geographic distribution patterns in cacao plantations in Ghana and Côte d'Ivoire were investigated through field surveillance, PCR detection assays, sequencing of positive amplicons, and phylogeographic clustering. Cumulatively, the detection efficiency of the tested CSSD primer sets that were targeting the movement protein domain of the virus ranged from 0.15% (CSSD-3 primer) to 66.91% (CSSD-1 primer) on all the symptomatic cacao leaf samples assessed. The identified CSSD species differed phylogenetically and overlapped in distribution, with the cacao swollen shoot Togo B virus (CSSTBV) (n = 588 sequences) being the most prevalent and widely distributed compared to the other CSSD species that were encountered in both countries. Geographically, the cacao swollen shoot CE virus (CSSCEV) species (n = 124 sequences) that was identified was largely restricted to the bordering regions of Ghana and Côte d'Ivoire. These results provide updated knowledge of the geographic distribution of the key CSSD species and their diagnostic efficiency and, thus, provide guidance in identifying locations for structured testing of cacao germplasm and optimal diagnostics for the predominant CSSD species in Ghana and Côte d'Ivoire.

Bioinformatic, genetic and molecular analysis of several badnavirus sequences integrated in the genomes of diverse cocoa (Theobroma cacao L.) germplasm

Endogenous viral elements (EVEs) are integrations of whole or partial viral genomes into the host genome, where they act as host alleles. They exist in a wide range of plant species including Theobroma cacao, the source of chocolate. Because of the international transfer of cacao germplasm, it is important to discriminate between the presence of these inserts and any episomal viruses that may be present in the material. This study was designed to survey a wide range of cacao germplasm, to assess the number, length, orientation, and precise location of the inserts and to identify any effect on the transcription of the gene into which they are inserted. Using a combination of bioinformatic, genetic and molecular approaches, we cloned and sequenced a series of different inserts, including one full-length virus sequence. We also identified, for the first time, an inhibitory effect of the insert on the expression of host genes. Such information is of practical importance in determining the regulation of germplasm transfer and of fundamental relevance to aiding an understanding of the role that such inserts may have on the performance of the host plant.

A Previously Undescribed Polerovirus (Solemoviridae) Infecting Theobroma cacao Germplasm.

Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al., 2018). Virus-like symptoms consisting of discoloration, leaf distortion with downward rolling of leaves, and yellow speckling or mottling (Fig. S1), were observed in imported cacao germplasm at the USDA-ARS-SHRS cacao quarantine facilities in the fall of 2020. Total RNA was isolated from leaves collected from four symptomatic plants using silica RNA extraction method (Rott and Jelkmann, 2001). Ribosomal RNA (rRNA)-depleted RNA samples were used for cDNA library construction, followed by high throughput sequencing on Illumina® NovaSeq 6000 platform (Novogene Corp., Sacramento, CA). Quality-filtered, 150-bp paired-ended reads (2,601,293-3,104,474) were assembled de novo using SPAdes v.3.14.1 (Nurk et al., 2013). The contigs (200,799 to 276,851) were queried against the NCBI virus reference sequence (RefSeq) database using the discontiguous megablast algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). The resultant contigs (n=1,344) were 150-nt to 1463-nt in length (k-mer coverage from 6.3x to 26,721.7x) and shared their highest nucleotide (nt) identity with potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae), at 69.1%-72.8%. The contigs pooled from the four samples were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds against the RefSeq database indicated the best matches were to thirteen other polerovirus species, with top hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), having similarity scores of 66.2% and 100% respectively. The 15 scaffolds matched to the 5' terminal end, ORF1-2, ORF3, ORF4 and ORF3-5 of the different polerovirus genomes. For confirmatory sequencing, total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA), followed by RT-PCR amplification with general polerovirus primers PoconF/PoconcpR (Xiang et al., 2008) expected to yield an amplicon of ~1,400-bp located at the 3' end of the RNA-dependent, RNA polymerase (RdRp), including the complete coat protein (CP) and movement protein (MP) genes. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI) and sequenced bi-directionally by Sanger sequencing (Eton Bio, Research Triangle Park, NC). BLASTn analysis of the polerovirus-like nt sequences (GenBank accession nos. (ON745771-ON745774) indicated the closest relatives were potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), at 71% and 73%, respectively. The CP amino acid (aa) sequence shared the greatest similarity to cereal yellow dwarf virus RPV (NP_840023), at 53%, and the MP aa sequence shared the greatest aa similarity to wheat yellow leaf dwarf virus-GPV (YP_003029842), at 38%. These results provide robust support for the association of a previously undescribed polerovirus with symptomatic cacao trees, herein named, cacao leafroll virus (Solemoviridae; Polerovirus). Although Koch's postulates have not been completed to confirm causality, the presence of this virus in cacao germplasm undermine efforts to distribute pathogen-free germplasm and may pose a risk to cacao production in trees established from virus-infected plant material. To our knowledge, this is the first report of a polerovirus infecting cacao trees. All trees of these accessions at the quarantined facility in Miami, FL have been destroyed.

Growth and Nutritional Responses of Juvenile Wild and Domesticated Cacao Genotypes to Soil Acidity

Cacao is an important tropical crop and requires high-fertility soils for better growth and productivity; nevertheless, soils where this crop is grown are, in general, acidic and low in fertility. Therefore, germplasm with tolerance to soil acidity is desirable for cacao genetic improvement. The objective of this study was to evaluate cacao germplasm for early growth, nutrient concentration, and potential tolerance to soil acidity. A greenhouse experiment was conducted to evaluate 60 cacao genotypes with diverse geographic origins. Cacao seedlings were grown for six months in acid soil with and without lime. Growth parameters and the total concentration of N, P, K, Ca, Mg, Fe, Cu, Mn, and Zn were measured in shoots after harvest. Our results indicate that the best early growth predictors of acid soil tolerance are the number of leaves and root area. N, Ca, Mg, and K uptake may have a potential role in tolerance to soil acidity. Finally, the results revealed a large difference among cacao genotypes in terms of their responses to acid soil stress, which led to the selection of ten genotypes: CCN-51, PH-21, CCN-10, PAS-91, ICT-1087, ICS-95, UF-667, TSH-565, PH-144, ICT-1189 that are potentially tolerant to soil acidity and could be used for breeding acid soil-tolerant cacao varieties.

Agromorphological characterization of cacao (Theobroma cacao L.) accessions from the germplasm bank of the National Institute of Agrarian Innovation, Peru

Agromorphological characterization of cacao accessions in Peru is currently an important tool in the conservation and genetic improvement of cacao germplasm. The objective of this study was to carry out the morphological and agronomic characterization of 113 cacao accessions from the Huarangopampa germplasm bank. Tree, leaf, flower, fruit and seed descriptors were used. The data collected were processed by descriptive statistics using multivariate techniques. Five groups were formed according to similar characteristics. The accessions of group 1 are vigorous trees with an pod index of 19.27 pods/kg of seeds; the groups that presented better differential characteristics were group 2 with erect tree architecture, intermediate vigorousness, purple seed color and pod index of 20.07 pods/kg of seeds and group 3, which had the highest number of accessions with the lowest pod index of 18.77 pods/kg of seeds, besides being vigorous trees and having purple seeds. On the other hand, group 4 presented a particular characteristic of white seed color and high pod indexes with 22.11 pods/kg of seeds. Finally, group 5 accessions were characterized by intermediate tree architecture and vigor with an pod index of 21.3 pods/kg of seeds. The morphoagronomic characterization constitutes a first advance in the identification of cacaos with potential for genetic improvement and advances in the Peruvian chocolate industry.

First Report of Diaporthe tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico.

Worldwide cacao pod rot is a devastating disease of Theobroma cacao, infected cacao pods turn necrotic reducing yield up to 30%. From July 2020 to August 2021, a survey was conducted at the USDA-ARS cacao germplasm collection located at Mayaguez, Puerto Rico. Incidence of cacao pod rot was 73.9%, observed in 142 of the 196 accessions sampled. The disease was observed at different stages of pod development (small, green, mature pods, and dry mummified large pods). Diseased tissue from three cacao pods (1 mm2) per each cacao accession was surface disinfested by immersion in 70% ethanol for one minute, rinsed with sterile-distilled-water and plated on potato dextrose agar (PDA) amended with 250 mg/L ampicillin and 60 mg/L streptomycin. After 30 days of incubation at 25°C, seven isolates developing white fast-growing colonies with black-globose pycnidia were observed. All isolates produced hyaline, one-celled, biguttulate, and cylindrical and rounded at the apex α conidia of 5.1 to 7.3 µm × 2.5 to 3.0 µm in size and were identified as Diaporthe spp. (Gomes et al. 2013; Crous et al. 2015). To determine the species identity, seven isolates were sequenced of the internal transcribed spacer (ITS), sections of β-tubulin (BT) and translation elongation factor 1 alpha (EF1-α) and compared using the BLASTn with Diaporthe spp. type specimens deposited in NCBI GenBank. ITS, BT and EF1-α sequences of Phomocac16, Phomcac17, Phomcac18 and Phomcac21 isolates (GenBank accession nos. OL353698 to OL353701, OL412430 to OL412433, and OL412437 to OL412440 for ITS, BT and EF1-α, respectively) were grouped to the holotype BRIP 62248a (Bootstrap BS=100) of Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan. The other three isolates (Phomcac8P1, Phomcac8P3 and Phomcac8P4) were grouped to the ex-type (CBS 101339) of Diaporthe pseudomangiferae R.R. Gomes, Glienke & Crous, ITS, BT and EF1-α sequences of (GenBank accessions nos. OL353702 to OL353704, OL412434 to OL412436, and OL412441 to OL412443, for ITS, BT and EF1-α, respectively). Pathogenicity tests were conducted using isolates Phomocac16, Phomcac17, Phomcac18 and Phomcac21 of D. tulliensis and isolates Phomcac8P1, Phomcac 8P3 and Phomcac8P4 of D. pseudomangiferae on five healthy detached green, yellow and red pods of the following cacao varieties: TARS27, ICS16, ICS1, GS29, UF601, SIAL56, Amelonado, SIAL98, EET94, ICS129 and GNV58. Cacao pods were wounded and inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown on PDA of each isolate and wrapped with parafilm. Untreated controls were inoculated with PDA disks only. Fruits were kept in a humid chamber for 8 days at 25°C. Tests were repeated twice. Eight days after inoculation with D. tulliensis and D. pseudomangiferae, all cacao pods turned dark brown, untreated controls showed no symptoms of pod rot, and no fungi were isolated from tissue. Both species, D. tulliensis and D. pseudomangiferae were reisolated from their respective diseased tissues fulfilling Koch's postulates. Diaporthe tulliensis has been reported from rotted stem ends of cacao pods in Australia (Crous et al. 2015), and D. pseudomangiferae was reported in a shipment of cacao seed pods in California; however, pathogenicity tests were not conducted at either location. In California D. pseudomangiferae is considered a quarantine pathogen with a temporary Q rating (Chitambar 2017). To our knowledge, this is the first report of D. tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico. Knowing the identity and incidence of these new cacao pathogens is the first step for developing specific control measures and potential sources for resistance to cacao pod rot caused by Diaporthe spp.

Phylogenetic diversity of cacao (Theobroma cacao L.) genotypes in Colombia

AbstractTheobroma cacao L. (cacao) is an important tropical crop used to produce chocolate. Evolutionary relationships between cultivated and wild cacao genotypes and their genetic diversity are poorly understood. Exploring phylogenetic diversity and spatial patterns of both cultivated and crop wild relatives can improve the knowledge of the evolutionary history of a crop, giving insights into its cultivation, breeding programmes and conservation. This study identifies biodiversity priority areas in Colombia by calculating phylogenetic diversity indices using a set of 87 single nucleotide polymorphism markers. These were sourced from 279 genotypes conserved in the Corporación Colombiana de Investigación Agropecuaria (Agrosavia) germplasm collection. The Caribbean and North Andes areas exhibited the highest phylogenetic diversity and significantly high relative phylogenetic diversity. We propose that those regions where wild cacao occurs should be prioritized as conservation areas. Besides, cacao lineages that have recently diverged and are present in Arauca, Huila and Nariño areas, with significantly low relative phylogenetic diversity, should be prioritized for breeding programmes. The Amazonia genotypes were closer to the root of the phylogenetic tree, suggesting an older origin than those found in the Andes region. Our study highlights the importance of using T. cacao germplasm from the Amazonia region as a priority to recover relict diversity in breeding programmes and broaden the gene pool of modern cultivated cacao.

Phenotypic Characterization of Fine-Aroma Cocoa from Northeastern Peru

The phenotypic characterization of cacao (Theobroma cacao L.) plays an important role in the generation of information for the conservation of cacao germplasm. The objective of this study is to characterize phenotypically 146 ecotypes of fine-aroma native cacao (FFNC) from northeastern Peru that were collected from 280 to 1265 metres above sea level. Morphological descriptors of fruits and seeds, sensory characteristics, and productivity descriptors were used. The data obtained were analyzed using descriptive statistics with pie charts, distribution histograms, and multiple correspondence analysis. The results showed that 76.7% of the cocoa ecotypes had green immature fruits, 73% showed slight roughness on the surface of the fruit, 54% showed an intermediate thickness of the fruit wall, and 90% had the appearance of pairs of equidistant ridges. Regarding seed characteristics, 71% showed purplish cotyledons, with a high presence of floral and fruity notes and low levels of bitterness and astringency. Likewise, 52% of the fruits and 64% of the seeds were long. More importantly, cocoa beans needed to produce between 14 and 16 pods to obtain one kilogram of dry cocoa, which reflects a good level of productivity. Finally, there was a positive relationship between elevation levels and the presence of fine-flavoured native cocoa, i.e., the greatest diversity of native cocoa with floral and fruity notes was found above 501 metres above sea level.

KERAGAMAN KARAKTERISTIK MORFOLOGIS TANAMAN KAKAO (Theobroma cacao L.) DI KECAMATAN HARAU KABUPATEN LIMA PULUH KOTA

Generally, the cacao farmers in Harau, Lima Puluh Kota District cultivating the cacao crops that unclear identity of its seed and there is also a high level of cacao diversity. Therefore the objectives of this study were to observe the morphological characters of cacao in Harau, Lima Puluh Kota District, and collecting data on the morphological characteristics of it as initial information on cacao germplasm development. This research was conducted by a survey, obtained information from the Agriculture Department, interviewed the cacao farmers, and collected data directly in the field. The method used in this research was a purposive sampling. The observed cacao crops as samples were taken from 3 Nagari in Harau, Lima Puluh Kota District, namely Nagari Taram with 4 cacao variants, Nagari Bukik Limbuku with 4 cacao variants, and Nagari Solok Bio-bio with 3 cacao variants. The obtained data were analyzed by NTSYS Program Ver. 02 which aims to know the diversities among them. The similarity of the phenotypes with a coefficient of 33-58 % and the variation is 42-67 %. Phenotypic variability of cacao crops on qualitative characters was a high value (41-85 %), and on quantitative characters was a moderate variability value ranges 3-50 %.

Traditional varieties of cacao (<em>Theobroma cacao</em>) in Madagascar: their origin and dispersal revealed by SNP markers

Cacao (<em>Theobroma cacao</em> L.) is an important Neotropical crop originating in South America and dispersed by European explorers, arriving in Madagascar in the late 19^th century. Although Madagascar is an important producer of cocoa for the premium chocolate market, the varietal composition and genetic diversity in cacao germplasm from Madagascar, especially in traditional cacao farms, remains unknown. A total of 190 cacao accessions, including 40 farmer accessions collected from traditional cacao farms in Madagascar, and 150 accessions representing seven reference cacao populations, were analyzed using Single Nucleotide Polymorphism (SNP) markers. Multivariate analysis and Bayesian stratification resulted in the clustering of the 40 farmer accessions into three groups: Criollo, Amelonado and Trinitario. These three traditional varieties were commonly cultivated in tropical America in the 18^th century, but most of them have been replaced by improved varieties. The present study demonstrated that Madagascar is distinctive in that all three traditional cacao varieties, Criollo, Amelonado and Trinitario, are still maintained on-farm for cocoa production, as in Mesoamerica and the Caribbean several hundred years ago. Results from the present study are significant in terms of understanding the early dispersal of cacao from tropical America and Asia to Africa, in addition to the well-documented route from Brazil to São Tomé &amp; Príncipe. The results also provide new information for planning future conservation and utilization of cacao germplasm in Madagascar.

Molecular Characterization of a Cacao Germplasm Collection Maintained in Yunnan, China Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important crop, since it provides the raw material for the multibillion dollar global chocolate industry. Yunnan Province is in the far southwestern region of China where tropical agriculture makes up a significant part of the provincial GDP. There is great potential for cacao production in Yunnan, given the rapid increase of chocolate consumption in China. Understanding the genetic background of available germplasm is essential for effective genebank management and crop genetic improvement. The objectives of this investigation were to assess the genetic identity of the cacao germplasm maintained in Yunnan, China; and to quantify the level of genetic diversity in comparison with available diversity in international collections. Using Single Nucleotide Polymorphism (SNP) markers, we genotyped the cacao collection maintained in the Xishuangbanna Tropical Botanical Garden in Yunnan. Multivariate analysis and Bayesian assignment test, using 150 reference cacao accessions, showed that these introduced cacao germplasm accessions have a narrow genetic background and were mostly comprised of Amelonado hybrids. Of the 88 Yunnan accessions, 59% of the parentage is contributed by Amelonado and 17% by two Upper Amazon Forastero populations. The study demonstrated that these introduced cacao germplasm accessions have a narrow genetic background thus lacking multiple sources of resistance to major cacao diseases. The present results therefore suggest that new introductions are needed to enrich genetic diversity in this collection. An improved germplasm collection would serve as a foundation for broader on-farm diversity for cacao cultivation in Yunnan, as well as in other tropical regions of China.

Genetic diversity and ancestry of cacao (Theobroma cacao L.) in Dominica revealed by single nucleotide polymorphism markers.

Cacao (Theobroma cacao L.), an introduced tree crop in Dominica, is important for foreign exchange earnings from fine or flavour cocoa. The genetic structure of farmed cacao in Dominica was examined to identify varieties for conservation, breeding, and propagation to improve their cocoa industry. Cacao trees (156) from 73 sites over seven geographical regions were genotyped at 192 single nucleotide polymorphism (SNP) markers. Identity, regional differentiation, phylogenetic, multi-variate, ancestry, and core collection analyses were performed. Farmed cacao germplasm had moderate gene diversity (He = 0.320 ± 0.005) from generally unique trees, but cocoa growing regions were genetically similar. Synonymous matching (16.3%) showed that some clonal material was supplied to farmers. Cacao trees were mainly mixed from Amelonado, Criollo, Iquitos, Contamana, and Marañon ancestries, with predominantly Amelonado-Criollo hybrids. Criollo ancestry, linked to fine or flavour cocoa, was found at more than 30% in 28 unique trees. Forty-five trees, containing the SNP diversity of cacao in Dominica, are recommended as a core germplasm collection. This study identifies promising trees for improving cocoa quality; provides genetic evidence that community, regional, or country-wide pooling would not compromise the exclusive fine or flavour cocoa industry; and discusses other implications towards improving the Dominican cocoa industry.

Widely distributed variation in tolerance to Phytophthora palmivora in four genetic groups of cacao

The tropical tree Theobroma cacao is the source of chocolate, and its seeds are a major export from many producing countries in Central and South America, Africa, and Asia. Every year, 30–40% of pre-harvest yield is lost due to disease damage. Host plant resistance is the most efficient and environmentally friendly approach for disease management. Historically, cacao germplasm resources have been underutilized in efforts to introduce novel sources of disease tolerance into breeding programs. Maintenance of cacao germplasm also relies on clonally propagated live collections, as cacao seeds do not exhibit dormancy and cannot be stored for more than a few weeks. In this study, we use a 90 SNP array to verify genetic identity of a set of clones in the International Cocoa Collection at CATIE, Costa Rica, and assign the clones into known genetic groups. We also used a detached leaf inoculation technique to measure the susceptibility of 60 genotypes to Phytophthora palmivora, a major cacao pathogen with global importance. We identified 24 genotypes with disease tolerance statistically similar to a standard tolerant variety (SCA6) and another 24 which performed similarly to a standard susceptible variety (ICS1). Our results indicate that each of the four included genetic groups show variability for quantitative resistance to P. palmivora. These results provide a foundation for future genomic and transcriptomic analysis of disease tolerance and susceptibility in the field at CATIE and provide guidelines for breeders searching for novel sources of tolerance that can be introduced into breeding programs.

Genetic identity and origin of “Piura Porcelana”—a fine-flavored traditional variety of cacao (Theoborma cacao) from the Peruvian Amazon

Cacao (Theobroma cacao L.) is a tropical rainforest tree that is indigenous to the Amazon region of South America. The myriad of river basins present in the Upper Amazon are where the largest diversity of cacao populations is found. Although it is generally accepted that cacao was first cultivated in Mesoamerica, there is evidence for domestication in South America, the center of origin. “Piura Porcelana” is a traditional cacao variety from northern Peru that is cultivated for the gourmet chocolate market. The demand for fine-flavored cacao used in high-end chocolates and the corresponding premium prices paid for distinct cacao flavors provide economic incentives for the use and conservation of these traditional varieties. In the present study, we compared the genetic identity of Piura Porcelana cacao with 11 known cacao germplasm groups existing in the ex situ cacao genebanks, along with the living wild populations from the Santiago and Morona river valleys in northern Peru. The results of SNP analysis showed that Piura Porcelana shares the same general population membership with the “Nacional” cacao from Ecuador. However, Piura Porcelana significantly differed from Nacional (Fst = 0.185, P < 0.001), suggesting its unique genetic status. The high genetic similarity between Piura Porcelana and newly collected wild cacao suggested that the wild progenitors of Pirua Porcelana were from these river valleys, and this traditional variety could have been the result of independent domestication in northern Peru. These results provide new evidence of cacao domestication in the Amazon and will contribute to the sustainable use and conservation of these cacao genetic resources.

The physiological responses of cacao to the environment and the implications for climate change resilience. A review

Cacao (Theobroma cacao L.) is a tropical perennial crop which is of great economic importance to the confectionary industry and to the economies of many countries of the humid tropics where it is grown. Some recent studies have suggested that climate change could severely impact cacao production in West Africa. It is essential to incorporate our understanding of the physiology and genetic variation within cacao germplasm when discussing the implications of climate change on cacao productivity and developing strategies for climate resilience in cacao production. Here, we review the current research on the physiological responses of cacao to various climate factors. Our main findings are as follows: (1) water limitation causes significant yield reduction in cacao, but genotypic variation in sensitivity is evident; (2) in the field, cacao experiences higher temperatures than is often reported in the literature; (3) the complexity of the cacao/shade tree interaction can lead to contradictory results; (4) elevated CO2 may alleviate some negative effects of climate change; (5) implementation of mitigation strategies can help reduce environmental stress; and (6) significant gaps in the research need addressing to accelerate the development of climate resilience. Harnessing the significant genetic variation apparent within cacao germplasm is essential to develop modern varieties capable of high yields in non-optimal conditions. Mitigation strategies will also be essential, but to use shading to best effect shade tree selection is crucial to avoid resource competition. Cacao is often described as being sensitive to climate change, but genetic variation, adaptive responses, appropriate mitigation strategies and interactive climate effects should all be considered when predicting the future of cacao production. Incorporating these physiological responses to various environmental conditions and developing a deeper understanding of the processes underlying these responses will help to accelerate the development of a more resource use efficient tree ensuring sustainable production into the future.

Molecular Characterization of Cacao (Theobroma cacao) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important commodity in Jamaica. Knowledge of the genetic diversity of Jamaican cacao germplasm is essential for their conservation and management. In spite of cacao’s economic importance in Jamaica, the crop is under studied, therefore limiting sound decisions toward improving productivity. Assessment of germplasm and on-farm genetic diversity is required to assist selecting superior genotypes to propagate and distribute across the island, as well as to use them as parental clones in breeding programs. Using 94 single nucleotide polymorphism (SNP) markers, 140 Jamaican cacao samples from two germplasm collections and a farmer’s estate along with 150 reference samples were analyzed. The principal coordinate analysis demonstrated that the majority of the Jamaican cacao selections were hybrids derived from five original germplasm groups, including Criollo, Amelonado and three Upper Amazon Forastero groups. Among the Upper Amazon groups, the Bayesian clustering analysis revealed that the Parinari (PA) ancestral lineage contributed the most (29.9%) to the Jamaican cacao germplasm. The germplasm collections showed greater diversity in terms of ancestral contributions compared to the farmer’s estate. However, the genetic differentiation between the three collecting sites was small (Fst = 0.036), indicating that samples collected from the three sites were derived from a common pool of germplasm. The current study supports the historical records and clarified the ancestry of Jamaican cacao. Although the majority of the cacao genetic groups were observed in the Jamaican cacao collections, several diversity gaps were found in both germplasm collections and in the farmer’s estate, especially germplasm with disease resistance to cacao frosty pod rot that was recently found in Jamaica.

Genetic identity and diversity of Nigerian cacao genebank collections verified by single nucleotide polymorphisms (SNPs): a guide to field genebank management and utilization

Nigeria is the sixth largest cacao producer in the world. Field performance and quality of cacao hybrid families is largely dependent on the genetic integrity of parental clones obtained in field genebank collections. However, information on the impact of mislabeling on seed garden output in Nigeria is lacking. Using 63 single nucleotide polymorphism (SNP) markers, we analyzed 1457 cacao trees sampled from seven major field genebank plots in Nigeria to assess the genetic integrity in Nigerian cacao germplasm. The procedure of multilocus matching with known reference clones revealed up to 78% mislabeling in recently introduced international germplasm. A high rate of mislabeling was also revealed in the West African local selections and breeding lines, using Bayesian assignment test. The problem of mislabeling has been attributed to errors from the sources of introduction, pre-planting labeling errors, and rootstocks overtaking budded scions due to poor field management. The analysis of genetic diversity revealed a good representation of the available cacao germplasm groups in Nigerian field genebanks, indicating that the genetic base of Nigeria cacao germplasm has been significantly widened through germplasm introductions. However, only a small proportion of the available germplasm in the genebank have been utilized for variety development. This study proved the utility of SNP markers for cleaning up the genebanks and reducing offtypes; thereby providing a strong basis for improving the accuracy and efficiency in cacao genebank management and breeding, as well as for mobilizing improved varieties to cacao farmers in Nigeria.

Assessing hidden parentage and genetic integrity of the "United Fruit Clones" of cacao (Theobroma cacao) from Costa Rica using SNP markers.

The international cacao collection in CATIE, Costa Rica contains nearly 1200 accessions of cacao, mainly from the center of genetic diversity of this species. Among these accessions, the United Fruit clones (UF clones) were developed by the United Fruit Company in Costa Rica, and they represent one of the earliest groups of improved cacao germplasm in the world. Some of these UF clones have been used as key progenitors for breeding resistance/tolerance to Frosty Pod and Black Pod diseases in the Americas. Accurate information on the identity and background of these clones is important for their effective use in breeding. Using Single Nucleotide Polymorphism (SNP) markers, we genotyped 273 cacao germplasm accessions including 44 UF clones and 229 reference accessions. We verified the true-to-type identity of UF clones in the CATIE cacao collection and analyzed their population memberships using maximum-likelihood-based approaches. Three duplicate groups, representing approximately 30% of the UF clones, were identified. Both distance- and model-based clustering methods showed that the UF clones were mainly composed of Trinitario, ancient Nacional and hybrids between ancient Nacional and Amelonado. This result filled the information gap about the UF clones thus will improve their utilization for cacao breeding.