Raymond J. Schnell

Geographic and Genetic Population Differentiation of the Amazonian Chocolate Tree (Theobroma cacao L)

Numerous collecting expeditions of Theobroma cacao L. germplasm have been undertaken in Latin-America. However, most of this germplasm has not contributed to cacao improvement because its relationship to cultivated selections was poorly understood. Germplasm labeling errors have impeded breeding and confounded the interpretation of diversity analyses. To improve the understanding of the origin, classification, and population differentiation within the species, 1241 accessions covering a large geographic sampling were genotyped with 106 microsatellite markers. After discarding mislabeled samples, 10 genetic clusters, as opposed to the two genetic groups traditionally recognized within T. cacao, were found by applying Bayesian statistics. This leads us to propose a new classification of the cacao germplasm that will enhance its management. The results also provide new insights into the diversification of Amazon species in general, with the pattern of differentiation of the populations studied supporting the palaeoarches hypothesis of species diversification. The origin of the traditional cacao cultivars is also enlightened in this study.

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.

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.

Theobroma cacao L.: a genetic linkage map and quantitative trait loci analysis.

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 1∶1 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC1 generation using both single-point ANOVA and interval mapping. A minimum number of 2–4 QTLs (P<0.01) involved in the genetic expression of the traits studied was detected. Coincident map locations of a QTL for jorquette height and trunk diameter suggests the possibility of pleiotropic effects in cocoa for these traits. The combined estimated effects of the different mapped QTLs explained between 11.2% and 25.8% of the phenotypic variance observed in the BC1 population.

Correction: Phylogenetic Analysis of Seven WRKY Genes across the Palm Subtribe Attaleinae (Arecaceae) Identifies Syagrus as Sister Group of the Coconut

Background The Cocoseae is one of 13 tribes of Arecaceae subfam. Arecoideae, and contains a number of palms with significant economic importance, including the monotypic and pantropical Cocos nucifera L., the coconut, the origins of which have been one of the “abominable mysteries” of palm systematics for decades. Previous studies with predominantly plastid genes weakly supported American ancestry for the coconut but ambiguous sister relationships. In this paper, we use multiple single copy nuclear loci to address the phylogeny of the Cocoseae subtribe Attaleinae, and resolve the closest extant relative of the coconut. Methodology/Principal Findings We present the results of combined analysis of DNA sequences of seven WRKY transcription factor loci across 72 samples of Arecaceae tribe Cocoseae subtribe Attaleinae, representing all genera classified within the subtribe, and three outgroup taxa with maximum parsimony, maximum likelihood, and Bayesian approaches, producing highly congruent and well-resolved trees that robustly identify the genus Syagrus as sister to Cocos and resolve novel and well-supported relationships among the other genera of the Attaleinae. We also address incongruence among the gene trees with gene tree reconciliation analysis, and assign estimated ages to the nodes of our tree. Conclusions/Significance This study represents the as yet most extensive phylogenetic analyses of Cocoseae subtribe Attaleinae. We present a well-resolved and supported phylogeny of the subtribe that robustly indicates a sister relationship between Cocos and Syagrus. This is not only of biogeographic interest, but will also open fruitful avenues of inquiry regarding evolution of functional genes useful for crop improvement. Establishment of two major clades of American Attaleinae occurred in the Oligocene (ca. 37 MYBP) in Eastern Brazil. The divergence of Cocos from Syagrus is estimated at 35 MYBP. The biogeographic and morphological congruence that we see for clades resolved in the Attaleinae suggests that WRKY loci are informative markers for investigating the phylogenetic relationships of the palm family.

Phylogenetic Relationships and Biogeography within the Eurasian Clade of Amaryllidaceae Based on Plastid ndhF and nrDNA ITS Sequences: Lineage Sorting in a Reticulate Area?

Abstract The monophyletic Eurasian clade of Amaryllidaceae was analyzed using plastid ndhF and rDNA ITS sequences for 33 and 29 taxa, respectively; all genera were represented by at least one species. Both maximum parsimony and Bayesian analysis were used on each data set and the combined data. Both sequence matrices resolve the Central and East Asian tribe Lycorideae as sister to the Mediterranean-centered genera of the clade, and recognize two large subclades within the greater Mediterranean region: Galantheae, consisting of Acis, Galanthus and Leucojum; and Narcisseae (Narcissus and Sternbergia)/Pancratium. However, there are areas of incongruence between the ndhF and ITS trees. When three predominantly monotypic genera, Hannonia, Lapiedra, and Vagaria, centered in North Africa, are removed from the alignments, the two sequence matrices produce fully congruent topologies with increased support at many of the nodes, with ILD between partitions rising from P = 0.07 to 0.96. We hypothesize that lineage sorting took place after the divergence of Galantheae and Narcisseae/Pancratium from a common genepool with Hannonia, Lapiedra, and Vagaria retaining a mosaic of the ancestral haplotypes. We also performed dispersal-vicariance analysis to reconstruct biogeographic scenarios on several of the generic level phylogenies found with and without these three genera included, as well as on a species-level phylogeny of Galantheae. After the vicariant divergence of the Asian Lycorideae, North Africa and the Iberian Peninsula are the most likely areas of origin for the rest of the clade. The results of the dispersal-vicariance analysis are discussed in the context of the complex biogeographic history of the Mediterranean basin.

Rapid propagation of tuna (Opuntia ficus-indica) and plant establishment in soil

Explants from young joints of mature plants of tuna (Opuntia ficus-indica Mill.) were cultured on Murashige and Skoog (MS) medium containing 8.8 μM benzyladenine (BA) and 0.5 μM naphthaleneacetic acid (NAA). Shoots produced were utilized as secondary explants. Each shoot was cut longitudinally from apex to base into two explants, and some of these explants were cut transversely into proximal and distal explants. The size and number of shoots produced was affected by size and position of the explant within its source. The shoots were rooted in vitro or ex vitro and plants were successfully established in soil from both rooting methods.

In vitro shoot proliferation and propagation of guava (Psidium guajava L.) from germinated seedlings

Guava seeds were germinated on Murashige and Skoog (MS) medium with or without 8.8 μM benzyladenine (BA). BA increased the rate of germination and the number of lateral shoots (3.4 vs 1.2 per seedling). Stem nodes from these lateral shoots were cultured on proliferation media with 4.4 μM BA, and multiple shoots (3.5) were formed within 4 weeks of culture. Increasing the concentration of BA or the addition of naphthaleneacetic acid (NAA) did not affect shoot formation. Shoots produced from explants and lateral shoots from germinated seedlings were rooted in media containing activated charcoal (AC) or 9.8 μM indolebutyric acid (IBA). Shoots rooted with IBA had a higher rooting percentage (100% vs 75%) and a greater number of roots (5.5 vs 3.2) but the shoots were shorter (2.6 vs 3.4 cm) than when rooted in AC, and they required an additional 4 weeks of culture in media with AC to achieve shoot elongation. About 80% of the shoots with roots survived in the glasshouse and produced normal phenotypic plants.

Variable response of open-pollinated seedling progeny of avocado to Phytophthora root rot

Phytophthora root rot, caused byPhytophthora cinnamomi Rands, is the most important disease of avocado (Persea americana Miller). In an attempt to identify root rot-resistant rootstocks that could ultimately be used under conditions in southern Florida, we screened open-pollinated progeny of avocado from the National Germplasm Repository in Miami. From 1996 to 1998, a total of 2,355 seedlings from 51 accessions were examined in potting mix artificially infested withP. cinnamomi. Most seedlings developed severe root rot, but tolerance was observed in some families (i.e., progeny of certain accessions). Although the most susceptible families developed mean disease ratings of up to 97% root necrosis, mean ratings for the most tolerant families were less than 60%. There was also a strong relationship between the racial background of the female parent and the tolerance of seedlings. Seedlings of the West Indian race and hybrids between it and the Guatemalan race were significantly more tolerant than those from other parents (P< 0.05). Individuals in several families developed < 50% root necrosis, the arbitrary standard of tolerance in this study. Twelve families accounted for 82% (188 of 229) of the tolerant seedlings, and only two of these did not have a West Indian or Guatemalan × West Indian pedigree. Broad-sense heritability for PRR tolerance was 0.45. This is the first report on the inheritance of PRR tolerance in avocado and on the influence of genotype and racial pedigree under controlled conditions.

AFLP analysis of genetic diversity within a jackfruit germplasm collection

Twenty-six jackfruit accessions, one interspecific hybrid, champedak, and one breadfruit accession were analyzed using amplified fragment length polymorphic (AFLP) markers to determine the degree of genetic diversity within the Fairchild Tropical Garden (FTG) germplasm collection. Of the 30 primer pairs evaluated, 12 were identified for collection screening based on number and quality of polymorphic fragments. A total of 187 AFLP markers were scored using the 12 primer pairs, 92 (49.2%) being polymorphic. All accessions could be uniquely identified using the 12 primer pairs. Among the jackfruit accessions, similarity coefficients ranged from 0.567 to 0.950; the accessions also shared a large number of monomorphic fragments (54.9%). Cluster analysis and principal component analysis (PCA) grouped all of the jackfruit accessions with south-east Asian origins into one major cluster with little bootstrap support for groupings within the cluster. The Indian accessions were grouped in a different cluster, as did the hybrid and the breadfruit accession. The AFLP marker based analysis indicates that limited genetic diversity exists within this collection. These observations are in agreement with the phenotypic evaluation and suggest that new accessions be obtained from the center of origin for the species.