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Caffeine is the most consumed alkaloid stimulant in the world. It is synthesized through the activity of three known N -methyltransferase proteins. Here we are reporting on the 422-Mb chromosome-level assembly of the Coffea humblotiana genome, a wild and endangered, naturally caffeine-free, species from the Comoro archipelago. We predicted 32,874 genes and anchored 88.7% of the sequence onto the 11 chromosomes. Comparative analyses with the African Robusta coffee genome ( C. canephora ) revealed an extensive genome conservation, despite an estimated 11 million years of divergence and a broad diversity of genome sizes within the Coffea genus. In this genome, the absence of caffeine is likely due to the absence of the caffeine synthase gene which converts theobromine into caffeine through an illegitimate recombination mechanism. These findings pave the way for further characterization of caffeine-free species in the Coffea genus and will guide research towards naturally-decaffeinated coffee drinks for consumers.

The GCAs are new tetraploid interspecific hybrids developed in Madagascar from Coffea eugenioides, C. canephora and C. arabica. Selected GCA having genotype UF1023 contained 0.37% DW caffeine and no detectable theobromine in green beans. Low caffeine accumulation in GCA plants is due mainly to the low biosynthetic activity of purine alkaloids, possibly the extremely weak N-methyltransferase reactions in caffeine biosynthesis. No significant catabolic activity of caffeine was found in GCA-UF1023, in common with almost all coffee plants including C. arabica.

Background and AimsThe coffee genus (Coffea) comprises 124 species, and is indigenous to the Old World Tropics. Due to its immense economic importance, Coffea has been the focus of numerous genetic diversity studies, but despite this effort it remains insufficiently studied. In this study the genetic diversity and genetic structure of Coffea across Africa and the Indian Ocean islands is investigated.MethodsGenetic data were produced using 13 polymorphic nuclear microsatellite markers (simple sequence repeats, SSRs), including seven expressed sequence tag-SSRs, and the data were analysed using model- and non-model-based methods. The study includes a total of 728 individuals from 60 species.Key ResultsAcross Africa and the Indian Ocean islands Coffea comprises a closely related group of species with an overall pattern of genotypes running from west to east. Genetic structure was identified in accordance with pre-determined geographical regions and phylogenetic groups. There is a good relationship between morpho-taxonomic species delimitations and genetic units. Genetic diversity in African and Indian Ocean Coffea is high in terms of number of alleles detected, and Madagascar appears to represent a place of significant diversification in terms of allelic richness and species diversity.ConclusionsCross-species SSR transferability in African and Indian Ocean islands Coffea was very efficient. On the basis of the number of private alleles, diversification in East Africa and the Indian Ocean islands appears to be more recent than in West and West-Central Africa, although this general trend is complicated in Africa by the position of species belonging to lineages connecting the main geographical regions. The general pattern of phylogeography is not in agreement with an overall east to west (Mascarene, Madagascar, East Africa, West Africa) increase in genome size, the high proportion of shared alleles between the four regions or the high numbers of exclusive shared alleles between pairs or triplets of regions.

The Coffea genus, 124 described species, has a natural distribution spreading from inter-tropical Africa, to Western Indian Ocean Islands, India, Asia and up to Australasia. Two cultivated species, C. arabica and C. canephora, are intensively studied while, the breeding potential and the genome composition of all the wild species remained poorly uncharacterized. Here, we report the characterization and comparison of the highly repeated transposable elements content of 11 Coffea species representatives of the natural biogeographic distribution. A total of 994 Mb from 454 reads were produced with a genome coverage ranging between 3.2 and 15.7 %. The analyses showed that highly repeated transposable elements, mainly LTR retrotransposons (LTR-RT), represent between 32 and 53 % of Coffea genomes depending on their biogeographic location and genome size. Species from West and Central Africa (Eucoffea) contained the highest LTR-RT content but with no strong variation relative to their genome size. At the opposite, for the insular species (Mascarocoffea), a strong variation of LTR-RT was observed suggesting differential dynamics of these elements in this group. Two LTR-RT lineages, SIRE and Del were clearly differentially accumulated between African and insular species, suggesting these lineages were associated to the genome divergence of Coffea species in Africa. Altogether, the information obtained in this study improves our knowledge and brings new data on the composition, the evolution and the divergence of wild Coffea genomes.

The completion of the genome assembly for the economically important coffee plant Coffea canephora (Rubiaceae) has allowed the use of bioinformatic tools to identify and characterize a diverse array of transposable elements (TEs), which can be used in evolutionary studies of the genus. An overview of the copy number and location within the C. canephora genome of four TEs is presented. These are tested for their use as molecular markers to unravel the evolutionary history of the Millotii Complex, a group of six wild coffee (Coffea) species native to Madagascar. Two TEs from the Gypsy superfamily successfully recovered some species boundaries and geographic structure among samples, whereas a TE from the Copia superfamily did not. Notably, species occurring in evergreen moist forests of eastern and southeastern Madagascar were divergent with respect to species in other habitats and regions. Our results suggest that the peak of transpositional activity of the Gypsy and Copia TEs occurred, respectively, before and after the speciation events of the tested Madagascan species. We conclude that the utilization of active TEs has considerable potential to unravel the evolutionary history and delimitation of closely related Coffea species. However, the selection of TE needs to be experimentally tested, since each element has its own evolutionary history. Different TEs with similar copy number in a given species can render different dendrograms; thus copy number is not a good selection criterion to attain phylogenetic resolution.

Madagascar has 59 species of Coffea, of which 42 are listed as Critically Endangered, Endangered, or Vulnerable by criteria of the Red List Category system of the World Conservation Union. In an attempt to assess the conservation value of ex situ collections of Malagasy coffee species, a study was undertaken using the field genebank collections maintained at the Kianjavato Coffee Research Station. Three species were selected for this purpose, C. kianjavatensis, C. montis - sacri, and C. vatovavyensis , and for comparative purposes extant, in situ populations of the same species were studied. Parentage analyses of ex situ propagated offspring of C. kianjavatensis and C. montis - sacri were performed to assess if crossing with other Coffea species maintained in the field genebank is compromising the genetic integrity of the collection. For these three species, higher genetic diversity was observed in the ex situ populations compared to the in situ populations, highlighting the importance of preserving the plants currently in ex situ collections. Parentage analyses of seed-propagated offspring of C. kianjavatensis and C. montis - sacri revealed that cross contamination with pollen from other Coffea species in the ex situ field genebank is occurring. These results have significant implications for the conservation management of wild Coffea species and for the management of ex situ genebanks.

The genus Coffea, mainly native to Africa and to the Indian Ocean islands (Mascarocoffea), accounts for 124 species. Genome size data are available for 23 African species. The aim of this study was to assess the genome size of 44 Mascarocoffea species and to investigate possible association with species geographic distribution, stomata traits, and species relationships. 2 C values were measured using flow cytometry. A lyophilization procedure for leaves was tested. The 2 C nuclear DNA content of Mascarocoffea species ranged from 0.96 to 1.41 pg. Coffea mauritiana and Coffea humblotiana have the smallest genomes and Coffea millotii has the largest. Mean 2 C DNA for Mascarocoffea and Africa is 1.19 and 1.43 pg, respectively. The overall DNA values corresponded to two partially overlapped normal distributions: one harboring species from east Africa Mascarocoffea, the other harboring species from west/central Africa. Plotted on a geographical map according to the native origin of species, these values showed a gradient in Madagascar and Africa. Genome sizes increased following a north to southeast gradient in Madagascar and an east to west gradient in Africa. None, or only weak correlations were noted between genome size and stomata parameters. Genetically close species could be highly distinctive in their genome size while divergent species could be similarly sized. The non-random geographic distribution and habitat of species, and the absence of correlation between genome size and genetic relationships, suggest that during Coffea genome evolution, both DNA content increase and/or decrease occurred independently in Africa and in the Indian Ocean Islands.

The genetic diversity of endemic Madagascan Coffea species has not received any considerable attention, despite the high extinction threat facing most of the species on the island. In order to address this shortfall, we assessed and compared the genetic diversity and allelic richness of ex situ and in situ populations for four selected species, and 18 ex situ populations maintained at the Kianjavato Coffee Research Station. We also investigated the possibility of in situ and ex situ hybridization events for Madagascan coffee, both within and between species. For these purposes, we used 18 nuclear microsatellite markers, on a total of 37 Coffea populations (4 in situ and 33 ex situ) representing a total of 398 genotypes (96 in situ and 302 ex situ) for 23 species. Our study showed (1) strong differentiation between Madagascan Coffea species; (2) good differentiation between all the populations studied, including those from the same locality (even when the area of the locality was restricted); (3) good evidence for both in situ and ex situ hybridization, although in situ hybridization appeared to be limited; (4) much higher genetic diversity within in situ collections compared to ex situ collections; and (5) that, despite limitations, the Kianjavato Coffee Research Station collection is an extremely valuable resource of Madagascan coffee germplasm. Recommendations for in situ and ex situ germplasm conservation are discussed.

†Background and Aims The phenolic composition of Coffea leaves has barely been studied, and therefore this study conducts the first detailed survey, focusing on mangiferin and hydroxycinnamic acid esters (HCEs). †Methods Using HPLC, including a new technique allowing quantification of feruloylquinic acid together with mangiferin, and histochemical methods, mangiferin content and tissue localization were compared in leaves and fruits of C. pseudozanguebariae, C. arabica and C. canephora. The HCE and mangiferin content of leaves was evaluated for 23 species native to Africa or Madagascar. Using various statistical methods, datawere assessed in relation to distribution, ecology, phylogeny and use. †Key Results Seven of the 23 species accumulated mangiferin in their leaves. Mangiferin leaf-accumulating species also contain mangiferin in the fruits, but only in the outer (sporophytic) parts. In both leaves and fruit, mangiferin accumulation decreases with ageing. A relationship between mangiferin accumulation and UV levels is posited, owing to localization with photosynthetic tissues, and systematic distribution in high altitude clades and species with high altitude representatives. Analyses of mangiferin and HCE content showed that there are significant differences between species, and that samples can be grouped into species, with few exceptions. These data also provide independent support for various Coffea lineages, as proposed by molecular phylogenetic analyses. Sampling of the hybrids C. arabica and C. heterocalyx cf. indicates that mangiferin and HCE accumulation may be under independent parental influence. †Conclusions This survey of the phenolic composition in Coffea leaves shows that mangiferin and HCE accumulation corresponds to lineage recognition and species delimitation, respectively. Knowledge of the spectrum of phenolic accumulation within species and populations could be of considerable significance for adaptation to specific environments. The potential health benefits of coffee-leaf tea, and beverages and masticatory products made from the fleshy parts of Coffea fruits, are supported by our phenolic quantification.

Madagascar has 59 described species of Coffea, of which 42 are listed as critically endangered, endangered, or vulnerable by the criteria of the Red List Category system of the World Conservation Union (IUCN). The littoral forest of Madagascar is a distinctive type of humid evergreen forest restricted to unconsolidated sand located within a few kilometers of the Indian Ocean, now persisting only as small fragments with ca. 10 % of its original range remaining. In an attempt to understand the genetic diversity of Madagascan coffee species, we studied ex situ and in situ populations of Coffea commersoniana, an endemic species of the littoral forests of southeastern Madagascar and soon to be impacted by mining activities in that region. The in situ populations studied showed higher genetic diversity than the ex situ population. The genetic partitioning among the two in situ populations of C. commersoniana was high enough to necessitate keeping the two populations separate for restoration purposes. Based on these findings, recommendations for conservation management (in situ and ex situ) are made.