Genetic diversity, conservation, and utilization of Theobroma cacao L.: genetic resources in the Dominican Republic

Cacao (Theobroma cacao L.) is a significant agricultural commodity in the Dominican Republic, which ranks 11th in the world for cacao exports. To estimate genetic diversity, determine genetic identity, and identify any labeling errors, 14 SSR markers were employed to fingerprint 955 trees among cacao germplasm accessions and local farmer selections (LFS). Comparisons of homonymous plants across plots revealed a significant misidentification rate estimated to be 40.9 % for germplasm accessions and 17.4 % for LFS. The 14 SSRs amplified a total of 117 alleles with a mean allelic richness of 8.36 alleles per locus and average polymorphism information content (PIC) value of 0.67 for the germplasm collection. Similar levels of variation were detected among the LFS where a total of 113 alleles were amplified with a mean of 8.07 alleles per locus and PIC of 0.57. The observed heterozygosity (H_obs) was 0.67 for the germplasm collection and 0.60 for LFS. Based on population structure analysis 43.9 % of the germplasm accessions and 72.1 % of the LFS are predominantly of the Amelonado ancestry. Among these Amelonado, 51.7 % for the germplasm collection and 50.6 % for LFS corresponded to Trinitario hybrid lineage. Criollo ancestry was found in 7.6 and 9.5 % of the germplasm accessions and LFS, respectively. The Contamana, Nacional, and Iquitos backgrounds were also observed in both populations, but the Curaray background was only detected in the germplasm accessions. No Purús or Guiana ancestry was found in either of the populations. Overall, significant genetic diversity, which could be exploited in the Dominican Republic breeding and selection programs, was identified among the germplasm accessions and LFS.     

The Criollo cacao tree (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.): a review

In commercial terms, Criollo cacao trees (Theobroma cacao L.) are reputed to be the source of the commercial product (fermented and dried cocoa beans), which sells for the best price on the market. Nevertheless, the term “Criollo” has numerous meanings and interpretations depending on if it is used by commercial users or botanists, growers or breeders. Our review aims to specify which cocoas can justifiably carry the Criollo name. “Criollo” is a botanical subspecies of Theobroma cacao, i.e. Theobroma cacao subsp. cacao; however, the true Criollos form just one of the ten currently accepted genetic groups in the species. We thus provide an overview of genetic studies on the subject (published or not), along with what is currently known about “True Criollo” or “Ancient Criollo” cacao trees. In fact, there are few representatives in collections that are duly acknowledged to be true Criollos, particularly in the two International Cocoa Genebanks, where only seven clones are available. It is nonetheless certain that some true Criollos do exist in other collections but have not been formally identified (by genetic studies) as members of the Criollo genetic group. Likewise, some true Criollos, be they cultivated or subspontaneous, exist in Mexico and Central and South America (Venezuela and Colombia). However, certain clones called “modern Criollos”, which are closely related to the true Criollos but arise from hybridization with other genetic groups, are more common.     

Lipid composition of wild ecuadorian Theobroma subincanum Mart. seeds and comparison with two varieties of Theobroma cacao L

The present work analyzes the lipid fraction from seeds of wild Ecuadorian Theobroma subincanum and selected commercial varieties of Theobroma cacao from Mexico (var. Criollo) and Ecuador (var. Arriba). The lipid fraction was obtained from the seeds through supercritical fluid extraction and analysis performed by preparatory thin-layer chromatography followed by gas chromatography. The results revealed that in T. subincanum the triglycerides contain fatty acids with longer chains. The melting point and peroxide and saponifiable numbers were determined for each Theobroma sample. The results lead to the conclusion that T. subincanum would produce a poorer quality butter than T. cacao. Nevertheless, the results do point toward a significant commercial use of T. subincanum for low-profile products.     

Sources of Resistance to Phytophthora Pod Rot at the International CocoaGenebank, Trinidad

The International Cocoa Genebank, Trinidad (ICG,T) contains about 2000 cacao (Theobroma cacao L.) accessions. The purpose of the collection is to provide a source of genes for the genetic improvement of cacao through breeding. In fulfilling this objective, it is imperative that this collection be evaluated to identify major sources of genes for the genetic improvement of important agronomic–economic traits. In this study, 816 cacao accessions were evaluated for resistance to Phytophthora pod rot (black pod disease), which causes enormous loss of yield in cocoa production throughout the world.The study identifies promising resistant genotypes that could be exploited in cacao breeding programmes. It further compares the levels of resistance to Phytophthora pod rot among two cacao types (wild and cultivated types), three major groups (Forastero, Trinitario and Refractario) and 11 accession groups (B, AM, CL, ICS, IMC, JA, LP, MOQ, NA, PA and TRD) . The distribution of scores for the 816 accessions showed skewness towards the susceptible end of the disease rating scale, indicating that a large proportion (68.9%) of the sample was susceptible (disease rating 6–8) to Phytophthora pod rot. However, 12.9% of the sample population was found to be resistant (disease rating 1–3) and 18.2% moderately resistant (disease rating 4–5). Significant differences were observed between the wild and cultivated accessions and among the Forastero, Refractario and Trinitario groups. Marked differences were also observed among 11 accession groups, each of which was represented by at least 20 genotypes. Higher proportions of resistant (17.7%) and moderately resistant (22.6%) genotypes were observed in the wild accessions than in the cultivated varieties (9.4% resistant and 14.4% moderately resistant). The Forastero group, consisting of many wild accessions, was found to contain more resistant (18.0%) and moderately resistant (23.1%) genotypes than either the Trinitario (4.8% resistant and 13.6% moderately resistant) or Refractario (11.3% resistant and 15.4% moderately resistant). Among 11 accession groups, the largest percentage of resistant (24.2%) and moderately resistant (28.8%) genotypes were found in the PA (Forastero). Different accession groups had varying proportions of resistant and moderately resistant genotypes. This is not unexpected in an out-breeding crop such as cacao with a high level of heterogeneity. This finding reinforces the idea of a pre-breeding programme (germplasm enhancement) to accumulate resistance genes over several populations as a strategy for improving the genetic base of resistance in national cacao breeding programmes. One hundred and five promising resistant genotypes were identified among the 816 accessions evaluated. These accessions are potential sources of resistance genes for the development of high-yielding resistant varieties in cacao.     

Assessing the Genetic Diversity in the International Cocoa Genebank,Trinidad (ICG,T) using Isozyme Electrophoresis and RAPD

Random amplified polymorphic DNA (RAPD) and isozyme electrophoresis (IE) techniques were used to estimate the level of genetic diversity in a sample of cacao germplasm existing at the International Cocoa Genebank, Trinidad. Twenty-six cocoa populations represented by 459 cocoa genotypes were analysed using IE and 22 populations represented by 353 cocoa genotypes were analysed using RAPD. Despite few differences in the classification of the populations, both techniques revealed three major groups: the indigenous trees, the cultivated Trinitario and the cultivated trees from Ecuador. Two-thirds of the partitioned diversity were found within populations and one-third between the populations, with both techniques.     

Verification of identities in global cacao germplasm collections

The ex situ conservation of cacao genetic resources is in the form of field genebanks. Misidentified trees though, represent a serious problem to (i) curators, who need to capture genetic diversity, and (ii) users of germplasm material, who must be certain of the identities of the accessions for proper evaluation of their results. The presence of mislabelled trees was assessed from data compiled in the International Cocoa Germplasm Database (ICGD) 2000 v4.1CD-ROM, which contains the published records of cacao accessions in global holdings. Circumstantial evidence revealed that many germplasm collections, especially those in Brazil (CEPEC), Colombia (ICA), Costa Rica (CATIE), Côte d'Ivoire, Mexico, Malaysia, Togo, Trinidad (ICG,T) and Venezuela (EEC) probably possessed misidentified material. The severity of the problem ranged from 15–44 % and is suggested as an area for immediate resolution. Based upon the morphological data provided by the ICGD CD-ROM, several accessions were highly suspect, including 'EET 19 [ECU]', 'EET 48 [ECU]', 'EET 162 [ECU]', 'ICS 1', 'ICS 6', 'ICS 43', 'ICS 89', 'ICS 100', 'IMC 67', 'PA 121 [PER]', 'POUND 7 [POU]', 'SCA 6', 'SPA 9 [COL]', 'UF 29', 'UF 613' and 'UF 667'. However, the identification of mislabelled trees should depend ultimately on molecular analysis. Hence, the use of voucher specimens for verification is forwarded and the concept of a public domain database for microsatellite analyses of germplasm holdings is raised.     

Effect of zinc application on the dry matter yield uptake and distribution of zinc and other micronutrients in cocoa (Theobroma cacao. L.)

Abstract

The effects of application of zinc fertilizer on dry matter yield, uptake and distribution of zinc and other nutrients by Amazon and Amelonado cocoa cultivars grown in a soil of low zinc content in the greenhouse were investigated.

There was a differential response to zinc fertilization by the two cocoa cultivars. Maximum dry matter yields of Amazon and Amelonado were attained with 10 ppm Zn and 50 ppm Zn, respectively. Under similar experimental conditions Amelonado seedlings expressed zinc deficiency symptoms whereas Amazon did not.

Zinc concentrations in the leaves, stem and roots of both cuitivars did not give a good index of the zinc status of the crops. This was because of the existence of the “Piper‐Steenbjerg”; effect in that nil zinc rates often gave higher leaf concentration of the zinc than next higher rate. In general, the relative content of zinc followed the pattern; leaves > roots > stem with the Amazon cultivar containing more zinc than the Amelonado.

The distribution of absorbed Cu in the leaves, stem and roots did not differ in both varieties. Whereas Fe uptake was mostly concentrated in the roots, Mn absorbed was largely concentrated in the leaves of both varieties and only Mn uptake in the leaves of Amazon consistently increased with Zn application.

The differences in the uptake and distribution of nutrients between the two cocoa cultivars was attributed to differences in their ability to extract nutrients from the soil and in their requirements for metabolic processes.     

Conservation and use of genetic resources of cacao (Theobroma cacao L.) by gene banks and nurseries in six Latin American countries

Genetic Resources and Crop Evolution - Cacao (Theobroma cacao L.) is among the most important cash crops in tropical countries. The existing cacao genetic diversity represents a key resource to...     

Genetic diversity and spatial structure in a new distinct <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. population in Bolivia

Cacao (Theobroma cacao L.) is an important economic crop in the Bolivian Amazon. Bolivian farmers both cultivate cacao, and extract fruits from wild stands in the Beni River region and in valleys of the Andes foothills. The germplasm group traditionally used is presently referred to as “Cacao Nacional Boliviano” (CNB). Using DNA fingerprinting technology based on microsatellite markers, we genotyped 164 Bolivian cacao accessions, including both cultivated and wild CNB accessions sampled from the Amazonian regions of La Paz and Beni, and compared their SSR profiles with 78 reference Forastero accessions from Amazonian cacao populations, including germplasm from the Ucayali region of Peru. Results of multivariate ordination and analysis of molecular variance show that CNB cacao has a unique genetic profile that is significantly different from the known cacao germplasm groups in South America. The results also show that cultivated CNB and wild CNB populations in the Beni River share a similar genetic profile, suggesting that the cultivated CNB is of indigenous origin in Bolivia. The level of genetic diversity, measured by allele richness and gene diversity in the Bolivian cacao, is moderately high, but was significantly lower than gene diversity in the other Amazonian cacao populations. Significant spatial genetic structure was detected in the wild CNB population, using analysis of autocorrelation (rc = 0.232; P < 0.001) and Mantel tests (Rxy = 0.276; P < 0.001). This finding is also highly valuable to support in situ conservation and sustainable use of CNB genetic diversity in Bolivia.     

基于SNP标记的504份番茄种质资源遗传多样性分析

为深入了解番茄种质资源的遗传多样性,运用竞争性等位基因特异性PCR(KASP)技术,利用前期筛选出的60对多态性较高的单核苷酸多态性标记(SNP),对收集的504份番茄种质资源进行遗传多样性分析、聚类分析、主成分分析及群体结构分析。结果表明,60个SNP分子标记共检测到181个等位基因,基因多样性平均值为0.450,期望杂合率(He)平均值为0.069,多态性信息值(PIC)变化范围为0.171~0.583,平均值为0.381。在遗传距离为0.36时,504份番茄材料被划分为7个类群,各材料间的平均遗传距离为0.62;根据主成分分析结果将群体分为3个类群;基于SNP标记对参试材料进行群体结构分析,在K=3时,504份番茄种质资源被划分为3类。本研究筛选出的60对SNP标记的多态性为中度偏高,番茄种质资源遗传多样性较丰富,可为后续宁夏地区番茄的核心种质构建及种质资源的有效利用提供理论依据。     

Genetic diversity among varieties of Chia (Salvia hispanica L.)

Chia, Salvia hispanica L., was a staple crop in pre-Columbian Mesoamerica. Despite the great potential of the species as an oilseed crop, little research related to domesticated and wild varieties exists. A study was undertaken to assess genetic diversity among 38 wild and domesticated accessions of S. hispanica collected throughout Mesoamerica by using RAPD markers. Genetic diversity was higher among wild varieties (H _G= 0.15) than all domesticated varieties (H _G= 0.10) and modern commercial domesticated varieties (H _G= 0.02), suggesting a slight loss of diversity accompanying domestication and a near lack of diversity in modern commercial varieties. In addition, the preliminary results indicate that the center of genetic diversity is in the highlands of western Mexico.     

Diversity and differentiation of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> in Indonesia

Analysis of the genetic structure of Indonesian Oryza sativa and O. rufipogon using neighbour-joining trees based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers revealed that O. sativa in Indonesia is separated from O. rufipogon. Accessions of O. sativa in this study were differentiated into two major groups, indica and tropical japonica, excluding some varieties. SSR and SNP markers revealed the high value of differentiation (F _ST) and genetic distance (D) between indica and tropical japonica and we discovered four loci by SNP markers and one locus by SSR markers that play a role in differentiation between indica and tropical japonica. Interestingly, genetic diversity (H) in O. rufipogon was lower than that in O. sativa, however H in O. rufipogon was the highest and H in tropical japonica was the lowest when O. sativa was divided into two groups. Inbreeding coefficient (Fst) showed evidences that gene flow (Nm) between species and within species might be one of the mechanisms related to the diversification and differentiation of Indonesian rice germplasm by asymmetric pattern between species and within O. sativa as revealed by SSR and SNP markers. In addition, we found evidences on stabilizing selection in Indonesian rice germplasm and they might be the reasons why Indonesian rice germplasm did not differentiate due to source location of landrace. However, we found a weak relation between SSR and SNP markers probably due to highly polymorphic in SSR and the different properties of both markers.     

Nutritional survey of coffee and cacao groves in Sierra Leone

Abstract

A nutritional survey was conducted by collecting coffee and cacao leaf samples from various ecological zones in Sierra Leone and analysing for various elements_t Data obtained were compared with established leaf analysis standards used for assessing nutrient status of coffee and cacao leaves. Varying degrees of deficiencies of N, P, K, Mg, Mn and Cu were observed but excess levels of Fe were also recorded. Zinc and Ca levels were in the adequate range in coffee and cacao groves respectively.     

Antimutagenic activity of cacao: inhibitory effect of cacao liquor polyphenols on the mutagenic action of heterocyclic amines

We investigated the effect of polyphenols derived from cacao liquor on the mutagenic action of heterocyclic amines (HCAs) in vitro and ex vivo. In the Ames test, the cacao liquor polyphenols showed antimutagenic effects in bacteria treated with HCA in the presence of an S-9 mixture; however, they showed less efficacy than quercetin. On the other hand, the cacao liquor polyphenols showed potent antimutagenic activity in bacteria treated with activated forms of HCA, compared with quercetin. We also evaluated the effect of these compounds on enzymatic activation of HCA. They weakly suppressed the production of activated HCA. In the host-mediated assay in mice, a method used to estimate the potential carcinogenicity of chemicals ex vivo, oral administration of the cacao liquor polyphenols, reduced the number of colonies of revertant bacteria recovered from the liver. These data suggest that the cacao liquor polyphenols have an antimutagenic effect not only in vitro, but also ex vivo.     

Spatial and temporal dynamics in genetic diversity in upland rice and late millet (<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.) in The Gambia

Ambiguity exists about the level of genetic diversity represented by farmer crop varieties, how it develops over time and how it relates to the diversity comprised by formal varieties. As part of an interdisciplinary technological/sociological study on farmer management of gene flow, upland rice (Oryza sativa L.) and late millet (Pennisetum glaucum (L.) R.Br.) from The Gambia were investigated for morphological and molecular variation. The goal of these analyses was to obtain insight into the level of crop genetic diversity of farmer’s materials planted in several case study villages in The Gambia. For both crops, samples were collected from villages and various research institutes. Based on variety names, different rice and millet varieties were expected to be used in different villages. In fact, there was a large overlap in genetic diversity for both crops, masked by the use of synonyms. The considerable similarity in rice genetic diversity between villages most likely results from the exchange of varieties between farmers. For millet this seems the result of development of varieties from the same gene pool. Some farmer varieties of rice, however, are apparent hybrid forms between the species O. sativa and O. glaberrima Steud., and farmer varieties in general displayed higher levels of genetic diversity than formal varieties. This indicates that, for rice, genetic diversity develops in farmers’ fields and may have potential use in formal breeding programs.     

Diversity of pomegranate (Punica granatum L.) germplasm in Tunisia

Thirty pomegranate (Punica granatum L.) accessions were studied to determine the overall degree of polymorphism and to detect similarities among genotypes. PCA and cluster analysis showed a considerable phenotypic and genetic diversity in the local pomegranate germplasm. Some polyclonal varieties were identified and cases of homonymy were detected. The geographic origin was not a determinant criterion for cultivars clustering. Parameters with high discriminating values were those related to fruit size and color and juice characteristics. Factors affecting pomegranate germplasm diversity are discussed.     

High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase

A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol-water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry.