Osman A. Gutierrez

Assessing microsatellite linkage disequilibrium in wild, cultivated, and mapping populations of Theobroma cacao L. and its impact on association mapping

Linkage disequilibrium (LD) measured over the genomes of a species can provide important indications for how future association analyses should proceed. This information can be advantageous especially for slow-growing, perennial crops such as Theobroma cacao, where experimental crosses are inherently time-consuming and logistically expensive. While LD has been evaluated in cacao, previous work has been focused on relatively narrow genetic bases. We use microsatellite marker data collected from a uniquely diverse sample of individuals broadly covering both wild and cultivated varieties to gauge the LD present in the different cacao diversity groups and populations. We find that genome-wide LD decays far more rapidly in the wild and primitive diversity groups of cacao as compared to those representing cultivated varieties. The impact that such differences can have on association analyses is demonstrated using phenotypic data on pod color and genotypic data from two cacao populations with contrasting patterns of LD decay. Our results indicate that the more rapid LD decay in wild and primitive germplasm can lead to higher-resolution mapping intervals when compared to results from cultivated germplasm. Through simulations, we demonstrate how future association mapping analyses, comprising of cacao samples with a wild or primitive background, will likely exhibit lower LD and would be more suitable for fine-scale association mapping analyses. As many traits targeted by cacao breeders are found exclusively in wild and primitive germplasm, association mapping in wild cacao populations holds significant promise for cacao improvement through marker-assisted breeding and emphasize the need to further explore the natural diversity of Amazonian cacao.

Breeding for Disease Resistance in Cacao

Cacao production must increase in order to meet the projected rise in the demand for chocolate. Approximately one-third of global production is lost annually to diseases and insects. Four diseases account for the greatest losses worldwide: black pod, caused by four Phytophthora spp.; witches’ broom, caused by Moniliophthora perniciosa; cacao swollen shoot virus, caused by a member of the genus Badnavirus; and frosty pod, caused by Moniliophthora roreri. At the present time, only 30 % of material currently under cultivation is of improved varieties, therefore, there is an urgent need for the development of new, high-yielding, disease-resistant varieties. Sustainable production increases could be achieved if improved varieties were used by the farmers. Cacao breeding was started in Trinidad in the 1930s by F. J. Pound and within a few decades cacao research centers had been established in all the major cacao producing areas worldwide including West Africa and Southeast Asia. Pound and other researchers have made several expeditions to the Amazon to collect wild cacao germplasm. In addition to using the germplasm collected from the wild and farmers’ fields to find new sources of resistance genes, researchers have developed breeding programs that cross and select cacao genotypes in order to accumulate desirable genes for resistance, as well as good horticultural and quality traits. Recently, numerous molecular tools, including the genome sequences of two varieties of cacao, have been developed and/or made available to accelerate the breeding process. International private/public collaborations are in progress to identify candidate resistance genes, map these in the sequenced genomes, and develop molecular markers associated with these genes. Researchers will use these markers in genomics-assisted breeding programs to screen young cacao plants and select those with desirable traits.

Molecular characterization of previously elusive badnaviruses associated with symptomatic cacao in the New World

Suspected virus-like symptoms were observed in cacao plants in Trinidad during 1943, and the viruses associated with these symptoms were designated as strains A and B of cacao Trinidad virus (CTV). However, viral etiology has not been demonstrated for either phenotype. Total DNA was isolated from symptomatic cacao leaves exhibiting the CTV A and B phenotypes and subjected to Illumina HiSeq and Sanger DNA sequencing. Based on de novo assembly, two apparently full-length badnavirus genomes of 7,533 and 7,454 nucleotides (nt) were associated with CTV strain A and B, respectively. The Trinidad badnaviral genomes contained four open reading frames, three of which are characteristic of other known badnaviruses, and a fourth that is present in only some badnaviruses. Both badnaviral genomes harbored hallmark caulimovirus-like features, including a tRNAMet priming site, a TATA box, and a polyadenylation-like signal. Pairwise comparisons of the RT-RNase H region indicated that the Trinidad isolates share 57-71% nt sequence identity with other known badnaviruses. Based on the system for badnavirus species demarcation in which viruses with less than 80% nt sequence identity in the RT-RNase gene are considered members of separate species, these isolates represent two previously unidentified badnaviruses, herein named cacao mild mosaic virus and cacao yellow vein banding virus, making them the first cacao-infecting badnaviruses identified thus far in the Western Hemisphere.

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.

Population genomic analyses of the chocolate tree, Theobroma cacao L., provide insights into its domestication process

Domestication has had a strong impact on the development of modern societies. We sequenced 200 genomes of the chocolate plant Theobroma cacao L. to show for the first time to our knowledge that a single population, the Criollo population, underwent strong domestication ~3600 years ago (95% CI: 2481–13,806 years ago). We also show that during the process of domestication, there was strong selection for genes involved in the metabolism of the colored protectants anthocyanins and the stimulant theobromine, as well as disease resistance genes. Our analyses show that domesticated populations of T. cacao (Criollo) maintain a higher proportion of high-frequency deleterious mutations. We also show for the first time the negative consequences of the increased accumulation of deleterious mutations during domestication on the fitness of individuals (significant reduction in kilograms of beans per hectare per year as Criollo ancestry increases, as estimated from a GLM, P = 0.000425).Omar Cornejo et al. report a genomic analysis of 200 cacao plants (Theobroma cacao L.) representing more than 10 genetically distinct populations. They identify metabolic and disease resistance genes as contributing to the domestication of cacao and show that domesticated populations maintain a high proportion of deleterious mutations.

Genetic differentiation, races and interracial admixture in avocado (Persea americana Mill.), and Persea spp. evaluated using SSR markers

Avocado (Persea americana Mill.) is a subtropical domesticated fruit tree indigenous to Mesoamerica. It is a member of the Lauraceae family and is separated into three horticultural races (Guatemalan, Mexican, and West Indian) mainly corresponding to their ecological adaptation, botanical, and physiological traits. Main objectives of this study were to characterize the population structure, genetic diversity, and horticultural race of a total of 354 Persea spp. trees whose origin is as follow: 221 trees [P. americana, (218), P. nubigena (2) and P. krugii (1)] from the USDA-ARS-Subtropical Horticultural Research Station, Miami; 105 trees from the Fairchild Farm [P. americana (104) and P. schiedeana (1)], and 28 trees collected in Mexico [P. schiedeana (23) and P. americana (5)]. The complexity of their interracial admixture; as well as mislabeling frequency was also evaluated. Molecular marker analysis utilizing a set of 55 simple sequence repeat (SSR) markers amplified a total of 869 alleles with a mean number of alleles per locus of 15.8 and average polymorphism information content value of 0.71, indicating a high variability in the allele frequency for the collection. Significant deviations from Hardy–Weinberg equilibrium were identified after Bonferroni correction for a large number of loci (48; 87%) due to the presence of null alleles. The main source of variation for this population was found to be within individuals (66.84%), with 19.30% variation among populations, and 13.86% variation among individuals within populations. Moreover, population specific inbreeding indices (FIS) were calculated for West Indian, Guatemalan, and Mexican [(0.1918; p value 0.0000), (0.1879; p-value 0.0000), (0.0925; p-value 0.0022)], respectively. Bayesian analysis divided the individual genotypes into groups associated with the Guatemalan, Mexican, West Indian races; interracial admixture; complex hybrids and P. schiedeana species. Also, results of the multivariate clustering method (PCA) and genetic distance analyses calculated among all possible individual combinations within the SSR diversity data agreed with Bayesian or Structure analyses results. The 55 SSRs provided complete resolution of all individuals and the estimated mislabeling error was approximately 0.28%.

The proposed new species, cacao red vein virus, and three previously recognized badnavirus species are associated with cacao swollen shoot disease

BackgroundCacao swollen shoot virus (CSSV), Cacao swollen shoot CD virus (CSSCDV), and Cacao swollen shoot Togo A virus (CSSTAV) cause cacao swollen shoot disease (CSSD) in West Africa. During 2000–2003, leaf and shoot-swelling symptoms and rapid tree death were observed in cacao in Cote d’Ivoire and Ghana. Molecular tests showed positive infection in only ~50–60% of symptomatic trees, suggesting the possible emergence of an unknown badnavirus.MethodsThe DNA virome was determined from symptomatic cacao samples using Illumina-Hi Seq, and sequence accuracy was verified by Sanger sequencing. The resultant 14, and seven previously known, full-length badnaviral genomic and RT-RNase H sequences were analyzed by pairwise distance analysis to resolve species relationships, and by Maximum likelihood (ML) to reconstruct phylogenetic relationships. The viral coding and non-coding sequences, genome organization, and predicted conserved protein domains (CPDs) were identified and characterized at the species level.ResultsThe 21 CSSD-badnaviral genomes and RT-RNase H sequences shared 70–100% and 72–100% identity, respectively. The RT-RNase H analysis predicted four species, based on an ≥80% species cutoff. The ML genome sequence tree resolved three well-supported clades, with ≥70% bootstrap, whereas, the RT-RNase H phylogeny was poorly resolved, however, both trees grouped CSSD isolates within one large clade, including the newly discovered Cacao red vein virus (CRVV) proposed species. The genome arrangement of the four species consists of four, five, or six predicted open reading frames (ORFs), and the CPDs have similar architectures. By comparison, two New World cacao-infecting badnaviruses encode four ORFs, and harbor CPDs like the West African species.ConclusionsThree previously recognized West African cacao-infecting badnaviral species were identified, and a fourth, previously unidentified species, CRVV, is described for the first time. The CRVV is a suspect causal agent of the rapid decline phenotype, however Koch’s Postulates have not been proven. To reconcile viral evolutionary with epidemiology considerations, more detailed information about CSSD-genomic variability is essential. Also, the functional basis for the multiple genome arrangements and subtly distinct CPD architectures among cacao-infecting badnaviruses is poorly understood. New knowledge about functional relationships may help explain the diverse symptomatologies observed in affected cacao trees.