Juan Carlos Motamayor

Cacao domestication I: the origin of the cacao cultivated by the Mayas

Criollo cacao (Theobroma cacao ssp. cacao) was cultivated by the Mayas over 1500 years ago. It has been suggested that Criollo cacao originated in Central America and that it evolved independently from the cacao populations in the Amazon basin. Cacao populations from the Amazon basin are included in the second morphogeographic group: Forastero, and assigned to T. cacao ssp. sphaerocarpum. To gain further insight into the origin and genetic basis of Criollo cacao from Central America, RFLP and microsatellite analyses were performed on a sample that avoided mixing pure Criollo individuals with individuals classified as Criollo but which might have been introgressed with Forastero genes. We distinguished these two types of individuals as Ancient and Modern Criollo. In contrast to previous studies, Ancient Criollo individuals formerly classified as ‘wild’, were found to form a closely related group together with Ancient Criollo individuals from South America. The Ancient Criollo trees were also closer to Colombian-Ecuadorian Forastero individuals than these Colombian-Ecuadorian trees were to other South American Forastero individuals. RFLP and microsatellite analyses revealed a high level of homozygosity and significantly low genetic diversity within the Ancient Criollo group. The results suggest that the Ancient Criollo individuals represent the original Criollo group. The results also implies that this group does not represent a separate subspecies and that it probably originated from a few individuals in South America that may have been spread by man within Central America.

The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color

BackgroundTheobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders.ResultsWe describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation.ConclusionsWe report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits.

Cacao domestication II: progenitor germplasm of the Trinitario cacao cultivar

Cacao (Theobroma cacao L.) has been cultivated in Central America since pre-Columbian times. The type of cacao cultivated in this region was called Criollo; cacao populations from the Amazon basin were called Forastero. The type of Forastero most commonly cultivated until 1950 was named Amelonado. Historical data show Trinitario cacao to have originated in Trinidad, resulting from natural hybridisation between Criollo and Amelonado Forastero. Doubts persist on the source of the Amelonado Forastero involved in the origin of Trinitario; the Amelonado parent may have come from the Lower Amazon, the Orinoco or the Guyanas. Most of the cacao cultivated worldwide until 1950 consisted of Criollo, Trinitario and Amelonado. From the early 1950s, Forastero material collected in the Upper Amazon region during the 1930s and 1940s began to be employed in breeding programmes. To gain a better understanding of the origin and the genetic basis of the cacao cultivars exploited before the utilisation of germplasm collected in the Upper Amazon, a study was carried out using restriction fragment length polymorphism and microsatellite markers. Trinitario samples from 17 countries were analysed. With molecular markers, it was possible to clearly identify three main genotypes (represented by clones SP1, MAT1-6 and SIAL70) implicated in the origin of most Trinitario clones.

Genetic diversity and structure of farm and GenBank accessions of cacao (Theobroma cacao L.) in Cameroon revealed by microsatellite markers

The genetic diversity of 400 accessions collected in cacao farms, 95 GenBank, and 31 reference accessions was analyzed using the 12 microsatellite markers. The GenBank and reference accessions were subdivided into 12 accession groups (AG) that belong to the traditional cacao genetic groups (GG) Lower Amazon Forastero (LA), Upper Amazon Forastero (UA), Trinitario, and Criollo (Cr). The 12-microsatellite loci revealed a total of 125 alleles, 113 of which were present in the farm accession group (FA). The within and between group variation for all AGs accounted respectively for 81% and 19% of the total molecular variation. The average Fis for the FA was 0.15 suggesting a moderate level of inbreeding. Significant differences for the level of gene diversity were found between the farm (0.50), GenBank (0.42 to 0.62), and reference (0.10 to 0.60) AGs. Genetic differentiation among AGs was variable with Fst values varying between 0.14 and 0.57 for the different AGs. Analysis using a Bayesian model-based method showed the existence of a high level of admixture for the farm accessions group. The LA genes were most represented in the FA (54%), followed by UA (33%) and Cr (7%). The genes of LA were also the most represented in the GenBank (48%), followed by UA (24%) and Cr (14%). Only 14% and 6% of the genes of the GenBank and farm accessions, respectively, could not be attributed to any of the reference GGs. The results suggest the predominating presence of LA genes in the Cameroon farm accessions and a high level of admixture, with apparent presence of genes of more than three GGs in most accessions. The traditional Trinitario types appear to have almost disappeared from farmers fields. The admixture must be the result of hybridization and recombination of these genes from the different GGs in seed gardens and in farmers’ fields. The use of selected farm accessions will depend on the GG that it belongs to and also on their level of heterozygosity. Further implications of the results for breeding and for introduction of new germplasm into the Cameroon GenBank are discussed.

Development of single nucleotide polymorphism markers in Theobroma cacao and comparison to simple sequence repeat markers for genotyping of Cameroon clones

Single nucleotide polymorphism (SNP) markers are increasingly being used in crop breeding programs, slowly replacing simple sequence repeats (SSR) and other markers. SNPs provide many benefits over SSRs, including ease of analysis and unambiguous results across various platforms. We have identified and mapped SNP markers in the tropical tree crop Theobroma cacao, and here we compare SNPs to SSRs for the purpose of determining off-types in clonal collections. Clones are used as parents in breeding programs and the presence of mislabeled clones (off-types) can lead to the propagation of undesired traits and limit genetic gain from selection. Screening was performed on 186 trees representing 19 Theobroma cacao clones from the Institute of Agricultural Research for Development (IRAD) breeding program in Cameroon. Our objectives were to determine the correct clone genotypes and off-types using both SSR and SNP markers. SSR markers that amplify 11 highly polymorphic loci from six linkage groups and 13 SNP markers that amplify eight loci from seven linkage groups were used to genotype the 186 trees and the results from the two different marker types were compared. The SNP assay identified 98% of the off-types found via SSR screening. SNP markers spread across multiple linkage groups may serve as a more cost-effective and reliable method for off-type identification, especially in cacao-producing countries where the equipment necessary for SSR analysis may not be available.

Analysis of molecular diversity in Crinipellis perniciosa with AFLP markers

Crinipellis perniciosa causes a serious disease of cacao known as witches’ broom (WB). Heritable resistance to witches’ broom has been used in cacao improvement programs. ‘SCA6’ and ‘SCA12’ are highly resistant and are the most commonly used parents in the breeding schemes. However, SCA hybrids are not resistant to witches’ broom in all production areas. Presumably, different populations of C. perniciosa cause these variable responses. Amplified fragment length polymorphism (AFLP) markers were used to assess variation and population structure in this pathogen. We examined 40 isolates of C. perniciosa and one isolate of Melanotus subcuneiformis. Nine of 64 primer pairs produced consistent and informative DNA amplification, and were used to screen all isolates. Fifteen haplotypes (AFLP fingerprints) were detected with 186 polymorphic markers. Cluster analysis grouped isolates of the C biotype (pathogenic on cacao) from Bolivia, Brazil, Ecuador and Trinidad together in a major cluster that was distinct from isolates of the S biotype (pathogenic on solanaceous hosts) and M. subcuneiformis. Isolates of the C biotype were divided further into well supported, country-specific groups. Segregation of AFLP alleles was not observed among basidiospore isolates from the same basidiome, broom, tree or field, supporting previous reports that the fungus did not outcross. The results corroborated prior conclusions that C. perniciosa was probably introduced into the Bahia state of Brazil from the Amazon basin. Representative isolates from the genetically distinct groups that were revealed will be used to examine pathogenic specialization in C. perniciosa and differential responses that have been reported in ‘SCA6’-derived germplasm.

Identification and mapping of conserved ortholog set (COS) II sequences of cacao and their conversion to SNP markers for marker-assisted selection in Theobroma cacao and comparative genomics studies

Theobroma cacao (cacao) is a tree cultivated in the tropics around the world for its seeds that are the source of both chocolate and cocoa butter. Genetic marker development for marker-assisted selection (MAS) is critical for the success of cacao breeding for disease resistance and yield. To develop conserved ortholog set II (COSII) single-nucleotide polymorphism (SNP) markers for MAS in cacao, we have used three strategies and three types of cacao genetic and sequence data to identify and map 98 cacao COSII genes. The resources available at the time these studies were first undertaken dictated the strategy utilized. For the first strategy, SNPs were identified using cacao expressed sequence tags homologous to COSII sequences. Strategy II utilized a leaf transcriptome of cacao genotype “Matina 1–6” and Strategy III the genomic sequence of a 3-Mb region of “Matina 1–6” linkage group 5 associated with an important quantitative trait locus (QTL) for resistance to black pod. We have identified SNP markers for 83 of the 98 mapped COSII genes, and 19 of these SNP markers co-locate with QTLs. These COSII SNP markers, the first identified for cacao, will be used for genotyping and off-typing in cacao breeding programs and employed for genetic mapping and syntenic studies to trace co-location of genes regulating traits of importance between cacao and other species.

SSCP markers provide a useful alternative to microsatellites in genotyping and estimating genetic diversity in populations and germplasm collections of plant specialty crops

For well‐studied plant species with whole genome sequence or extensive EST data, SNP markers are the logical choice for both genotyping and whole genome association studies. However, SNP markers may not address the needs of researchers working on specialty crops with limited available genomic information. Microsatellite markers have been frequently employed due to their robustness, but marker development can be difficult and may result in few polymorphic markers. SSCP markers, such as microsatellites, are PCR‐based and scored by electrophoretic mobility but, because they are based on SNPs rather than length differences, occur more frequently and are easier to develop than microsatellites. We have examined how well correlated the estimation of genetic diversity and genetic distance are in a population or germplasm collection when measured by 13 highly polymorphic microsatellite markers or 20 SSCP markers. We observed a significant correlation in pairwise genetic distances of 82 individuals in an international cacao germplasm collection (Mantel test Rxy=0.59, p<0.0001 for 10 000 permutations). Both sets of markers could distinguish each individual in the population. These data provide strong support for the use of SSCP markers in the genotyping of plant species where development of microsatellites would be difficult or expensive.

Making a chocolate chip: development and evaluation of a 6K SNP array for Theobroma cacao

Theobroma cacao, the key ingredient in chocolate production, is one of the worlds most important tree fruit crops, with ∼4,000,000 metric tons produced across 50 countries. To move towards gene discovery and marker-assisted breeding in cacao, a single-nucleotide polymorphism (SNP) identification project was undertaken using RNAseq data from 16 diverse cacao cultivars. RNA sequences were aligned to the assembled transcriptome of the cultivar Matina 1-6, and 330,000 SNPs within coding regions were identified. From these SNPs, a subset of 6,000 high-quality SNPs were selected for inclusion on an Illumina Infinium SNP array: the Cacao6kSNP array. Using Cacao6KSNP array data from over 1,000 cacao samples, we demonstrate that our custom array produces a saturated genetic map and can be used to distinguish among even closely related genotypes. Our study enhances and expands the genetic resources available to the cacao research community, and provides the genome-scale set of tools that are critical for advancing breeding with molecular markers in an agricultural species with high genetic diversity.

Optimization of a SNP assay for genotyping Theobroma cacao under field conditions

The tropical tree crop Theobroma cacao L. is grown commercially for its beans, which are used in the production of cocoa butter and chocolate. Although the upper Amazon region is the center of origin for cacao, 70% of the world’s supply of cacao beans currently comes from small farms in West Africa. While cacao breeding programs in producer nations are the source of improved planting material, modern marker-based breeding is difficult to perform due to the lack of genotyping facilities in these countries. While DNA extraction can be routinely performed, the equipment needed to analyze simple sequence repeats (SSRs) is seldom available, forcing the outsourcing of genotyping to foreign laboratories and delaying the breeding process. We describe a 5′ nuclease (TaqMan)-based single nucleotide polymorphism (SNP) assay for genotyping cacao plants under conditions similar to those found in most cacao-producing areas. The assay was tested under field conditions by planting open pollinated seeds of seven pods from four different maternal plants. The resulting 171 seedlings were successfully genotyped with 18 SNP markers representing 12 loci. The ability to use temperature-stable reagents and rapid DNA extraction methods is also explored. Additionally, by examining the seedling genotypes for the SNP markers and 14 additional SSR markers, we investigated whether seeds in a pod are the result of single or multiple pollination events. This simple, effective method of genotyping cacao seedlings in the field should allow for more efficient resource management of seed gardens and is currently being implemented in Ghana.