Genetic Population Structure of Cacao Plantings within a Young Production Area in Nicaragua

Significant cocoa production in the municipality of Waslala, Nicaragua, began in 1961. Since the 1980s, its economic importance to rural smallholders increased, and the region now contributes more than 50% of national cocoa bean production. This research aimed to assist local farmers to develop production of high-value cocoa based on optimal use of cacao biodiversity. Using microsatellite markers, the allelic composition and genetic structure of cacao was assessed from 44 representative plantings and two unmanaged trees. The population at Waslala consists of only three putative founder genotype spectra (lineages). Two (B and R) were introduced during the past 50 years and occur in >95% of all trees sampled, indicating high rates of outcrossing. Based on intermediate allelic diversity, there was large farm-to-farm multilocus genotypic variation. GIS analysis revealed unequal distribution of the genotype spectra, with R being frequent within a 2 km corridor along roads, and B at more remote sites with lower precipitation. The third lineage, Y, was detected in the two forest trees. For explaining the spatial stratification of the genotype spectra, both human intervention and a combination of management and selection driven by environmental conditions, appear responsible. Genotypes of individual trees were highly diverse across plantings, thus enabling selection for farm-specific qualities. On-farm populations can currently be most clearly recognized by the degree of the contribution of the three genotype spectra. Of two possible strategies for future development of cacao in Waslala, i.e. introducing more unrelated germplasm, or working with existing on-site diversity, the latter seems most appropriate. Superior genotypes could be selected by their specific composite genotype spectra as soon as associations with desired quality traits are established, and clonally multiplied. The two Y trees from the forest share a single multilocus genotype, possibly representing the Mayan, ‘ancient Criollo’ cacao

Diversity of cacao trees in Waslala, Nicaragua: Associations between genotype spectra, product quality and yield potential

The sensory quality and the contents of quality-determining chemical compounds in unfermented and fermented cocoa from 100 cacao trees (individual genotypes) representing groups of nine genotype spectra (GG), grown at smallholder plantings in the municipality of Waslala, Nicaragua, were evaluated for two successive harvest periods. Cocoa samples were fermented using a technique mimicking recommended on-farm practices. The sensory cocoa quality was assessed by experienced tasters, and seven major chemical taste compounds were quantified by near infrared spectrometry (NIRS). The association of the nine, partially admixed, genotype spectra with the analytical and sensory quality parameters was tested. The individual parameters were analyzed as a function of the factors GG and harvest (including the date of fermentation), individual trees within a single GG were used as replications. In fermented cocoa, significant GG-specific differences were observed for methylxanthines, theobromine-to-caffeine (T/C) ratio, total fat, procyanidin B5 and epicatechin, as well as the sensory attributes global score, astringency, and dry fruit aroma, but differences related to harvest were also apparent. The potential cocoa yield was also highly determined by the individual GG, although there was significant tree-to-tree variation within every single GG. Non-fermented samples showed large harvest-to-harvest variation of their chemical composition, while differences between GG were insignificant. These results suggest that selection by the genetic background, represented here by groups of partially admixed genotype spectra, would be a useful strategy toward enhancing quality and yield of cocoa in Nicaragua. Selection by the GG within the local, genetically segregating populations of seedpropagated cacao, followed by clonal propagation of best-performing individuals of the selected GG could be a viable alternative to traditional propagation of cacao by seed from open pollination. Fast and gentle air-drying of the fermented beans and their permanent dry storage were an efficient and comparatively easy precondition for high cocoa quality. (Résumé d'auteur

Fermented cocoa from 100 individual trees (genotypes) representing 9 cacao Genotype Groups. Relative amounts of chemical compounds and scores of sensory attributes.

<p>Two-harvest averages by Genotype Group (GG) are indicated in the top part. GGs coded by colors according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054079#pone.0054079-Trognitz2" target="_blank">[3]</a> (B; blue, Y; yellow, O; orange, E; gray, G; green, S; steel gray, A; aqua, P; purple, R; red).</p><p>Bottom part of table; individual GGs are ranked for each character (in descending order from positive to negative contribution to high quality cocoa). The probability P (*; <0.05, **; <0.01, ***; <0.001) of a character as under control by the GG is indicated in the last line. To decide which GG represents the greatest potential (as a decision support in selection) one could consider those that occur most frequently among the (highlighted in bold-face) top three ranks. These would be GGs S and R, both occur 5 times.</p

Bar plots of the 15-SSR-genotype of 106 cacao trees from farms at Waslala that were used for assessment of cocoa quality and yield potential.

<p>A single bar represents a tree’s cumulative genotype consisting of fractions of ancestral genotype spectra. The in total nine genotype spectra were determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054079#pone.0054079-Trognitz2" target="_blank">[3]</a> via Bayesian inference of population structure. Color coding of individual genotype spectra according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054079#pone.0054079-Trognitz2" target="_blank">[3]</a>. S; steel gray, G; green, P; purple, O; orange, R; red, E; gray, A; aqua, B; blue, Y; yellow. Their most probable ancestors and traditional cocoa types include for S; lower Amazon Forastero, G, P, O, and R; upper Amazon Forastero, E; upper Amazon Forastero isolated in coastal Ecuador, A, B; Trinitario, and Y; Criollo from Mesoamerica. The trees were grouped by their dominating genotype spectra. Group names of genotypes are indicated prior to tree name (as an example, tree W358 belongs to the S genotype group).</p

Summary of analyses of variance, average total quantities of chemical compounds, and sensory quality scores of cocoa samples.

<p>A quality attribute was considered as a function of Genotype Group (GG), Harvest, GG-Harvest interaction, and Trees (as replicates within a GG). The level of significance of variance components (F-test) is indicated (***; P<0.001, **; P<0.01, *; P<0.05, n.s.; not significant). There were 9 Genotype Groups (GG) and 95 (in harvest 1, non-fermented and fermented), 87 (harvest 2, non-fermented), and 78 (fermented) samples from individual trees. Df; error degrees of freedom (in analysis of variance). Grand mean quantities of compounds and their minima and maxima are presented in percent of dry matter.</p

Plots of theobromine/caffeine (T/C) ratio (ordinate) vs. total caffeine content (abscissa) determined for samples of non-fermented cocoa.

<p>a; first harvest period 2008–9, b; second harvest (2009–11), c; two-harvest average.</p

Method of fermenting several small cocoa bean samples from individual trees under identical conditions of recommended traditional processing practice (“microfermentation technique”).

<p>A wooden fermentation box of 0.6×0.6×0.6-m dimensions is filled with a uniform 180-l batch of fresh beans. Up to eight meshes containing the individual samples are evenly distributed across two layers and perfectly embedded in the batch beans. Banana leaf is added as a cover and to provide optimal conditions for fermenting bacteria. The entire volume is rearranged manually every second day. Fermentation time uniformly was six days.</p

Empirically determined mean values (expressed as percent of air-dried cocoa matter) and their statistics, generated and used at CIRAD to predict the quantity of several biochemical compounds in cocoa samples via Near Infrared Spectroscopy (NIRS).

<p>n; number of replicate measurements, Mean; arithmetic mean of all n measurements, SD; standard deviation, SE; standard error, R; coefficient of determination, SECV; standard error of cross validation.</p

Pairwise correlations of analytical (F; fermented and N; non-fermented samples) and sensory data of fermented samples obtained on up to 172 individual observations collected over two harvests of cocoa from 100 trees of smallholder farms around Waslala, Nicaragua.

<p>Color and shading intensity indicate levels of significance of correlations. Theobromine/caffeine (T/C) ratio and theobromine and fat contents of fermented cocoa (highlighted by green background) were the most important characteristics as determined in the analytics (see text).</p

Potential cocoa yield (<8% water content) of nine Genotype Groups (GG) and their ranking and differentiation by comparison of multiple means (t-test).

1<p>GG; Genotype Group, compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054079#pone-0054079-g003" target="_blank">Figure 3</a>, Samplings; Number of observations made on the trees. The objective of 2 samplings per tree was not achieved for all trees. # Trees; Number of trees within a GG, Yield; Least-squares mean potential yield per tree (in kg). The potential maximum single-tree yield was estimated as (no fruits past cherelle wilt stage) * (no beans/fruit) * (bean weight). Bean weight was averaged on 100 dry (<8% water content) beans. t-Test; least significant difference of yield by GG determined by multiple t-Tests. Values with identical letters are not significantly different.</p