Characterization of microsatellites in Xanthosoma sagittifolium (Araceae) and cross-amplification in related species.

PREMISE OF THE STUDY: To investigate the genetic diversity of a root crop, Xanthosoma sagittifolium, and to facilitate germplasm conservation, microsatellite loci were developed and characterized by genotyping 39 accessions from different geographic origins. • METHODS AND RESULTS: Using a microsatellite-enriched library approach, 17 polymorphic microsatellite markers were identified and characterized. The number of alleles for each locus ranged from two to six. Observed and expected heterozygosities ranged from 0.00 to 0.97 and from 0.09 to 0.78, respectively. Additionally, cross-amplification of these microsatellite markers was tested successfully in other species of Xanthosoma and Caladium, with rates varying from 23.5% to 100%. • CONCLUSIONS: These results indicate the effectiveness of microsatellite loci developed for the characterization of X. sagittifolium genetic diversity. They are crucial for the future investigation of population dynamics and clonal identification and, therefore, for prioritizing germplasm conservation. They should also enable research on other related species.

Insight into the wild origin, migration and domestication history of the fine flavour Nacional Theobroma cacao L. variety from Ecuador.

Ecuador's economic history has been closely linked to Theobroma cacao L cultivation, and specifically to the native fine flavour Nacional cocoa variety. The original Nacional cocoa trees are presently in danger of extinction due to foreign germplasm introductions. In a previous work, a few non-introgressed Nacional types were identified as potential founders of the modern Ecuadorian cocoa population, but so far their origin could not be formally identified. In order to determine the putative centre of origin of Nacional and trace its domestication history, we used 80 simple sequence repeat (SSR) markers to analyse the relationships between these potential Nacional founders and 169 wild and cultivated cocoa accessions from South and Central America. The highest genetic similarity was observed between the Nacional pool and some wild genotypes from the southern Amazonian region of Ecuador, sampled along the Yacuambi, Nangaritza and Zamora rivers in Zamora Chinchipe province. This result was confirmed by a parentage analysis. Based on our results and on data about pre-Columbian civilization and Spanish colonization history of Ecuador, we determined, for the first time, the possible centre of origin and migration events of the Nacional variety from the Amazonian area until its arrival in the coastal provinces. As large unexplored forest areas still exist in the southern part of the Ecuadorian Amazonian region, our findings could provide clues as to where precious new genetic resources could be collected, and subsequently used to improve the flavour and disease resistance of modern Ecuadorian cocoa varieties.

Microsatellite markers for the Chameleon grasshopper (Kosciuscola tristis) (Orthoptera: Acrididae), an Australian Alpine Specialist.

A set of polymorphic loci was characterised using an enrichment library for the Australian alpine specialist, the chameleon grasshopper (Kosciuscola tristis), an atypical grasshopper known for its remarkable temperature-controlled colour change. The number of alleles per locus ranged from three to 20 and observed heterozygosity from 0.16 to 0.76. These are the first microsatellite markers for a non-endangered Australian alpine animal and will inform questions of gene flow across the sky islands of this unique and threatened region.

A reference microsatellite kit to assess for genetic diversity of Sorghum bicolor (Poaceae).

PREMISE OF THE STUDY: Discrepancies in terms of genotyping data are frequently observed when comparing simple sequence repeat (SSR) data sets across genotyping technologies and laboratories. This technical concern introduces biases that hamper any synthetic studies or comparison of genetic diversity between collections. To prevent this for Sorghum bicolor, we developed a control kit of 48 SSR markers. METHODS AND RESULTS: One hundred seventeen markers were selected along the genome to provide coverage across the length of all 10 sorghum linkage groups. They were tested for polymorphism and reproducibility across two laboratories (Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement [CIRAD], France, and International Crops Research Institute for the Semi-Arid Tropics [ICRISAT], India) using two commonly used genotyping technologies (polyacrylamide gel-based technology with LI-COR sequencing machines and capillary systems with ABI sequencing apparatus) with DNA samples from a diverse set of 48 S. bicolor accessions. CONCLUSIONS: A kit for diversity analysis (http://sat.cirad.fr/sat/sorghum_SSR_kit/) was developed. It contains information on 48 technically robust sorghum microsatellite markers and 10 DNA controls. It can further be used to calibrate sorghum SSR genotyping data acquired with different technologies and compare those to genetic diversity references.

Isolation and characterization of twelve polymorphic microsatellite Loci for the cocoa mirid bug Sahlbergella singularis.

Mirids are the primary pests affecting cocoa production in Africa, but no genetic studies have been conducted on these insects. Here we report the isolation and characterization of 12 polymorphic microsatellite loci for Sahlbergella singularis. A microsatellite-enriched genomic DNA library was developed and screened to identify marker loci. Twelve polymorphic loci were identified by screening 28 individuals collected from one presumed population in cocoa plantations in Southern Cameroon. The number of alleles ranged from 5 to 25, whereas the observed and the expected heterozygosities ranged from 0.179 to 0.786 and from 0.671 to 0.946, respectively. Tests showed significant deviations from HW equilibrium for four loci, but no linkage disequilibrium was detected at any of the loci. No cross-species amplification was observed in two other mirid pests in Africa.

Assessment of the genetic diversity of the Tunisian citrus rootstock germplasm.

BACKGROUND: Citrus represents a substantial income for farmers in the Mediterranean Basin. However, the Mediterranean citrus industry faces increasing biotic and abiotic constraints. Therefore the breeding and selection of new rootstocks are now of the utmost importance. In Tunisia, in addition to sour orange, the most widespread traditional rootstock of the Mediterranean area, other citrus rootstocks and well adapted to local environmental conditions, are traditionally used and should be important genetic resources for breeding. To characterize the diversity of Tunisian citrus rootstocks, two hundred and one local accessions belonging to four facultative apomictic species (C. aurantium, sour orange; C. sinensis, orange; C. limon, lemon; and C. aurantifolia, lime) were collected and genotyped using 20 nuclear SSR markers and four indel mitochondrial markers. Multi-locus genotypes (MLGs) were compared to references from French and Spanish collections. RESULTS: The differentiation of the four varietal groups was well-marked. The groups displayed a relatively high allelic diversity, primarily due to very high heterozygosity. Sixteen distinct MLGs were identified. Ten of these were noted in sour oranges. However, the majority of the analysed sour orange accessions corresponded with only two MLGs, differentiated by a single allele, likely due to a mutation. The most frequent MLG is shared with the reference sour oranges. No polymorphism was found within the sweet orange group. Two MLGs, differentiated by a single locus, were noted in lemon. The predominant MLG was shared with the reference lemons. Limes were represented by three genotypes. Two corresponded to the 'Mexican lime' and 'limonette de Marrakech' references. The MLG of 'Chiiri' lime was unique. CONCLUSIONS: The Tunisian citrus rootstock genetic diversity is predominantly due to high heterozygosity and differentiation between the four varietal groups. The phenotypic diversity within the varietal groups has resulted from multiple introductions, somatic mutations and rare sexual recombination events. Finally, this diversity study enabled the identification of a core sample of accessions for further physiological and agronomical evaluations. These core accessions will be integrated into citrus rootstock breeding programs for the Mediterranean Basin.

Development of nuclear microsatellite markers for the fonio, Digitaria exilis (Poaceae), an understudied West African cereal.

PREMISE OF THE STUDY: We developed nuclear microsatellite primers to explore the genetic diversity, population genetic structure, and evolutionary history of the fonio (Digitaria exilis), an understudied cereal cultivated in West Africa. METHODS AND RESULTS: We used a microsatellite-enriched library approach to isolate and characterize 38 nuclear primer pairs (31 di-, five tri-, and two tetranucleotide repeats), of which 21 were polymorphic and exhibited a clear pattern in 36 accessions from West Africa. The number of alleles per locus ranged from two to 22, with a mean of 4.71, and expected heterozygosity ranged from 0.03 to 0.93. CONCLUSIONS: The developed set of 21 polymorphic SSR markers will provide tools for population and evolutionary genetics studies of the cultivated fonio.

Specific patterns of genetic diversity among aromatic rice varieties in Myanmar.

BACKGROUND: After observing peculiar rice varieties in Myanmar, in terms of classification in varietal groups and of grain quality, we focused on Myanmar varieties and analyzed variations at 19 microsatellite loci as well as sequences of the aroma gene BADH2. RESULTS: Microsatellites were able to retrieve the well-established classification into Indica (isozyme group 1), Japonica (group 6, comprising temperate and tropical forms) and specific groups from the Himalayan foothills including some Aus varieties (group 2) and some aromatic varieties (group 5). They revealed a new cluster of accessions close to, but distinct from, non-Myanmar varieties in group 5. With reference to earlier terminology, we propose to distinguish a group "5A" including group 5 varieties from the Indian subcontinent (South and West Asia) and a group "5B" including most group 5 varieties from Myanmar. In Myanmar varieties, aroma was distributed in group 1 (Indica) and in group 5B. New BADH2 variants were found. Some accessions carried a 43 bp deletion in the 3' UTR that was not completely associated with aroma. Other accessions, all of group 5B, displayed a particular BADH2 allele with a 3 bp insertion and 100% association with aroma. CONCLUSION: With the new group and the new alleles found in Myanmar varieties, our study shows that the Himalayan foothills contain series of non-Indica and non-Japonica varietal types with novel variations for useful traits.

Multidisciplinary perspectives on banana (Musa spp.) domestication.

Original multidisciplinary research hereby clarifies the complex geodomestication pathways that generated the vast range of banana cultivars (cvs). Genetic analyses identify the wild ancestors of modern-day cvs and elucidate several key stages of domestication for different cv groups. Archaeology and linguistics shed light on the historical roles of people in the movement and cultivation of bananas from New Guinea to West Africa during the Holocene. The historical reconstruction of domestication processes is essential for breeding programs seeking to diversify and improve banana cvs for the future.

The genome of Theobroma cacao.

We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.

A saturated SSR/DArT linkage map of Musa acuminata addressing genome rearrangements among bananas.

BACKGROUND: The genus Musa is a large species complex which includes cultivars at diploid and triploid levels. These sterile and vegetatively propagated cultivars are based on the A genome from Musa acuminata, exclusively for sweet bananas such as Cavendish, or associated with the B genome (Musa balbisiana) in cooking bananas such as Plantain varieties. In M. acuminata cultivars, structural heterozygosity is thought to be one of the main causes of sterility, which is essential for obtaining seedless fruits but hampers breeding. Only partial genetic maps are presently available due to chromosomal rearrangements within the parents of the mapping populations. This causes large segregation distortions inducing pseudo-linkages and difficulties in ordering markers in the linkage groups. The present study aims at producing a saturated linkage map of M. acuminata, taking into account hypotheses on the structural heterozygosity of the parents. RESULTS: An F1 progeny of 180 individuals was obtained from a cross between two genetically distant accessions of M. acuminata, 'Borneo' and 'Pisang Lilin' (P. Lilin). Based on the gametic recombination of each parent, two parental maps composed of SSR and DArT markers were established. A significant proportion of the markers (21.7%) deviated (p < 0.05) from the expected Mendelian ratios. These skewed markers were distributed in different linkage groups for each parent. To solve some complex ordering of the markers on linkage groups, we associated tools such as tree-like graphic representations, recombination frequency statistics and cytogenetical studies to identify structural rearrangements and build parsimonious linkage group order. An illustration of such an approach is given for the P. Lilin parent. CONCLUSIONS: We propose a synthetic map with 11 linkage groups containing 489 markers (167 SSRs and 322 DArTs) covering 1197 cM. This first saturated map is proposed as a "reference Musa map" for further analyses. We also propose two complete parental maps with interpretations of structural rearrangements localized on the linkage groups. The structural heterozygosity in P. Lilin is hypothesized to result from a duplication likely accompanied by an inversion on another chromosome. This paper also illustrates a methodological approach, transferable to other species, to investigate the mapping of structural rearrangements and determine their consequences on marker segregation.

Development and multiplexing of microsatellite markers in the polyploid perennial herb, Menyanthes trifoliata (Menyanthaceae).

PREMISE OF THE STUDY: We developed microsatellite primers to investigate genetic diversity and population genetic structure of the endangered herb Menyanthes trifoliata. METHODS AND RESULTS: Using the microsatellite-enriched library method, we identified 10 primer pairs in M. trifoliata. The primers amplified nine di- and one tri-nucleotide repeats with 4-13 alleles per locus in two Belgian populations. CONCLUSIONS: The results indicate that these markers offer an appropriate amount of variation to investigate genetic diversity, pollen dispersal (through paternity inference), and other conservation issues.

Development and assessment of Diversity Arrays Technology for high-throughput DNA analyses in Musa.

Diversity Arrays Technology (DArT) is a DNA hybridisation-based molecular marker technique that can detect simultaneously variation at numerous genomic loci without sequence information. This efficiency makes it a potential tool for a quick and powerful assessment of the structure of germplasm collections. This article demonstrates the usefulness of DArT markers for genetic diversity analyses of Musa spp. genotypes. We developed four complexity reduction methods to generate DArT genomic representations and we tested their performance using 48 reference Musa genotypes. For these four complexity reduction methods, DArT markers displayed high polymorphism information content. We selected the two methods which generated the most polymorphic genomic representations (PstI/BstNI 16.8%, PstI/TaqI 16.1%) to analyze a panel of 168 Musa genotypes from two of the most important field collections of Musa in the world: Cirad (Neufchateau, Guadeloupe), and IITA (Ibadan, Nigeria). Since most edible cultivars are derived from two wild species, Musa acuminata (A genome) and Musa balbisiana (B genome), the study is restricted mostly to accessions of these two species and those derived from them. The genomic origin of the markers can help resolving the pedigree of valuable genotypes of unknown origin. A total of 836 markers were identified and used for genotyping. Ten percent of them were specific to the A genome and enabled targeting this genome portion in relatedness analysis among diverse ploidy constitutions. DArT markers revealed genetic relationships among Musa genotype consistent with those provided by the other markers technologies, but at a significantly higher resolution and speed and reduced cost.

Towards the understanding of the cocoa transcriptome: Production and analysis of an exhaustive dataset of ESTs of Theobroma cacao L. generated from various tissues and under various conditions.

BACKGROUND: Theobroma cacao L., is a tree originated from the tropical rainforest of South America. It is one of the major cash crops for many tropical countries. T. cacao is mainly produced on smallholdings, providing resources for 14 million farmers. Disease resistance and T. cacao quality improvement are two important challenges for all actors of cocoa and chocolate production. T. cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses and quality improvement, nutritional and organoleptic, is also important for consumers. An international collaboration was formed to develop an EST genomic resource database for cacao. RESULTS: Fifty-six cDNA libraries were constructed from different organs, different genotypes and different environmental conditions. A total of 149,650 valid EST sequences were generated corresponding to 48,594 unigenes, 12,692 contigs and 35,902 singletons. A total of 29,849 unigenes shared significant homology with public sequences from other species.Gene Ontology (GO) annotation was applied to distribute the ESTs among the main GO categories.A specific information system (ESTtik) was constructed to process, store and manage this EST collection allowing the user to query a database.To check the representativeness of our EST collection, we looked for the genes known to be involved in two different metabolic pathways extensively studied in other plant species and important for T. cacao qualities: the flavonoid and the terpene pathways. Most of the enzymes described in other crops for these two metabolic pathways were found in our EST collection.A large collection of new genetic markers was provided by this ESTs collection. CONCLUSION: This EST collection displays a good representation of the T. cacao transcriptome, suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from these ESTs. It will provide numerous genetic markers that will allow the construction of a high density gene map of T. cacao. This EST collection represents a unique and important molecular resource for T. cacao study and improvement, facilitating the discovery of candidate genes for important T. cacao trait variation.

Patterns of introduction and diversification of Vanilla planifolia (Orchidaceae) in Reunion Island (Indian Ocean).

The cultivated species Vanilla planifolia is a typical example of a crop introduced from its area of origin (America) to new regions where natural pollinators are absent. Although the Vanilla cultivars are exclusively vegetatively propagated, a high degree of phenotypic variation is observed among the cultivars in their introduction areas such as Reunion Island. To test several hypotheses explaining this variation-different introduction events, somatic mutations and sexual reproduction (through manual pollination)-we used AFLP markers to elucidate the patterns of introduction of V. planifolia. Most of the accessions cultivated in the world were derived from a single accession, possibly the Mexican cultivar Mansa. The patterns of diversification of this cultivated species were also studied and compared with other cultivated (V. tahitensis) and wild (V. pompona and V. bahiana) species. Except for one particular phenotype ('Aiguille'), which may come from sexual reproduction, cultivated accessions exhibit very low levels of genetic diversity. They have evolved by the accumulation of point mutations through vegetative multiplication. The genetic diversity revealed could not explain the phenotypic diversity, which may be related to epigenetics or polyploidy. This new understanding of the basis of genetic diversity of vanilla may assist to improve management of genetic resources.

Adding value to cocoa (Theobroma cacao L.) germplasm information with domestication history and admixture mapping.

A sound understanding of crop history can provide the basis for deriving novel genetic information through admixture mapping. We confirmed this, by using characterization data from an international collection of cocoa, collected 25 years ago, and from a contemporary plantation. We focus on the trees derived from three centuries of admixture between Meso-American Criollo and South American Forastero genomes. In both cacao sets of individuals, linkage disequilibrium extended over long genetic distances along chromosome regions, as expected in populations derived from recent admixture. Based on loose genome scans, genomic regions involved in useful traits were identified. Fifteen genomic regions involved in seed and fruit weight variation were highlighted. They correspond to ten previously identified QTLs and five novel ones. Admixture mapping can help to add value to genetic resources and thus, help to encourage investment in their conservation.