Valérie Poncet

The coffee genome provides insight into the convergent evolution of caffeine biosynthesis

Coffee, tea, and chocolate converge Caffeine has evolved multiple times among plant species, but no one knows whether these events involved similar genes. Denoeud et al. sequenced the Coffea canephora (coffee) genome and identified a conserved gene order (see the Perspective by Zamir). Although this species underwent fewer genome duplications than related species, the relevant caffeine genes experienced tandem duplications that expanded their numbers within this species. Scientists have seen similar but independent expansions in distantly related species of tea and cacao, suggesting that caffeine might have played an adaptive role in coffee evolution. Science, this issue p. 1181; see also p. 1124 The genetic origins of coffee’s constituents reveal intriguing links to cacao and tea. Coffee is a valuable beverage crop due to its characteristic flavor, aroma, and the stimulating effects of caffeine. We generated a high-quality draft genome of the species Coffea canephora, which displays a conserved chromosomal gene order among asterid angiosperms. Although it shows no sign of the whole-genome triplication identified in Solanaceae species such as tomato, the genome includes several species-specific gene family expansions, among them N-methyltransferases (NMTs) involved in caffeine production, defense-related genes, and alkaloid and flavonoid enzymes involved in secondary compound synthesis. Comparative analyses of caffeine NMTs demonstrate that these genes expanded through sequential tandem duplications independently of genes from cacao and tea, suggesting that caffeine in eudicots is of polyphyletic origin.

Phylogeography and niche modelling of the relict plant Amborella trichopoda (Amborellaceae) reveal multiple Pleistocene refugia in New Caledonia

Amborella trichopoda Baill. (Amborellaceae, Amborellales), the sole living member of the sister group to all other extant angiosperms, is endemic to New Caledonia. We addressed the intraspecific phylogeography of Amborella by investigating whether its present population genetic structure could be related to its current and past habitats. We found moderate range‐wide genetic diversity based on nuclear microsatellite data and detected four well‐differentiated, geographically distinct genetic groups using Bayesian clustering analyses. We modelled the ecological niche of Amborella based on the current climatic and environmental conditions. The predictive ability of the model was very good throughout the Central East mainland zone, but Amborella was predicted in the northern part of the island where this plant has not been reported. Furthermore, no significant barrier was detected based on habitat suitability that could explain the genetic differentiation across the area. Conversely, we found that the main genetic clusters could be related to the distribution of the suitable habitat at the last glacial maximum (LGM, c. 21 000 years BP), when Amborella experienced a dramatic 96.5% reduction in suitable area. At least two lineages survived in distinct putative refugia located in the Massif des Lèvres and in the vicinity of Mount Aoupinié. Our findings finally confirmed the importance of LGM rainforest refugia in shaping the current intra‐ and interspecific diversity in New Caledonian plants and revealed the possibility of an as yet unreported refugium. The combination of niche modelling and population genetics thereby offered novel insight into the biogeographical history of an emblematic taxon.

Micro‐collinearity and genome evolution in the vicinity of an ethylene receptor gene of cultivated diploid and allotetraploid coffee species (Coffea)

Arabica coffee (Coffea arabica L.) is a self-compatible perennial allotetraploid species (2n=4x=44), whereas Robusta coffee (C. canephora L.) is a self-incompatible perennial diploid species (2n=2x=22). C. arabica (C(a) C(a) E(a) E(a) ) is derived from a spontaneous hybridization between two closely related diploid coffee species, C. canephora (CC) and C. eugenioides (EE). To investigate the patterns and degree of DNA sequence divergence between the Arabica and Robusta coffee genomes, we identified orthologous bacterial artificial chromosomes (BACs) from C. arabica and C. canephora, and compared their sequences to trace their evolutionary history. Although a high level of sequence similarity was found between BACs from C. arabica and C. canephora, numerous chromosomal rearrangements were detected, including inversions, deletions and insertions. DNA sequence identity between C. arabica and C. canephora orthologous BACs ranged from 93.4% (between E(a) and C(a) ) to 94.6% (between C(a) and C). Analysis of eight orthologous gene pairs resulted in estimated ages of divergence between 0.046 and 0.665 million years, indicating a recent origin of the allotetraploid species C. arabica. Analysis of transposable elements revealed differential insertion events that contributed to the size increase in the C(a) sub-genome compared to its diploid relative. In particular, we showed that insertion of a Ty1-copia LTR retrotransposon occurred specifically in C. arabica, probably shortly after allopolyploid formation. The two sub-genomes of C. arabica, C(a) and E(a) , showed sufficient sequence differences, and a whole-genome shotgun approach could be suitable for sequencing the allotetraploid genome of C. arabica.

Microcollinearity in an ethylene receptor coding gene region of the Coffea canephora genome is extensively conserved with Vitis vinifera and other distant dicotyledonous sequenced genomes

BackgroundCoffea canephora, also called Robusta, belongs to the Rubiaceae, the fourth largest angiosperm family. This diploid species (2x = 2n = 22) has a fairly small genome size of ≈ 690 Mb and despite its extreme economic importance, particularly for developing countries, knowledge on the genome composition, structure and evolution remain very limited. Here, we report the 160 kb of the first C. canephora Bacterial Artificial Chromosome (BAC) clone ever sequenced and its fine analysis.ResultsThis clone contains the CcEIN4 gene, encoding an ethylene receptor, and twenty other predicted genes showing a high gene density of one gene per 7.8 kb. Most of them display perfect matches with C. canephora expressed sequence tags or show transcriptional activities through PCR amplifications on cDNA libraries. Twenty-three transposable elements, mainly Class II transposon derivatives, were identified at this locus. Most of these Class II elements are Miniature Inverted-repeat Transposable Elements (MITE) known to be closely associated with plant genes. This BAC composition gives a pattern similar to those found in gene rich regions of Solanum lycopersicum and Medicago truncatula genomes indicating that the CcEIN4 regions may belong to a gene rich region in the C. canephora genome. Comparative sequence analysis indicated an extensive conservation between C. canephora and most of the reference dicotyledonous genomes studied in this work, such as tomato (S. lycopersicum), grapevine (V. vinifera), barrel medic M. truncatula, black cottonwood (Populus trichocarpa) and Arabidopsis thaliana. The higher degree of microcollinearity was found between C. canephora and V. vinifera, which belong respectively to the Asterids and Rosids, two clades that diverged more than 114 million years ago.ConclusionThis study provides a first glimpse of C. canephora genome composition and evolution. Our data revealed a remarkable conservation of the microcollinearity between C. canephora and V. vinifera and a high conservation with other distant dicotyledonous reference genomes. Altogether, these results provide valuable information to identify candidate genes in C. canephora genome and serve as a foundation to establish strategies for whole genome sequencing. Future large-scale sequence comparison between C. canephora and reference sequenced genomes will help in understanding the evolutionary history of dicotyledonous plants.

Advances in Coffea Genomics

Abstract Coffee is the second most valuable commodity exported by developing countries. The Coffea genus comprises over 103 species but coffee production uses only two species throughout the tropics: Coffea canephora, which is self-sterile and diploid and better known as Robusta, and C. arabica, which is self-fertile and tetraploid. With the arrival of new analytical technologies and the start of genome sequencing projects, it was clearly time to review the state of the art of coffee genetics and genomics. In the first part of this chapter, we present the main results concerning genetic diversity and phylogeny – the most advanced fields – based on large molecular marker sets, such as random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), intersimple sequence repeat (ISSR), single sequence repeats (SSRs), or conserved orthologue set (COS), which are mainly polymerase chain reaction (PCR) based. These markers also enable the construction of genetic maps and the identification of quantitative trait loci (QTLs) for both morphological and biochemical traits. In the second part, after reviewing current knowledge on variation in coffee genome size and insights into cytogenetics, we focus on currently available genomic resources and web facilities. Large sets of expressed sequences tags (ESTs) and bacterial artificial chromosome (BAC) libraries for both C. canephora and C. arabica have been obtained along with information on genes and specific metabolic pathways. In the final section, we describe recently designed tools and their ultimate goal, which is to facilitate the sequencing, assembly and annotation of the first Coffea genome. We are at the gate of a new era of scientific approaches to coffee that should lead to a better understanding of phylogenetic relationships and genome evolution within the genus. Finally, taken together, this information should help develop improved varieties to meet the new challenges represented by ongoing radical changes in the environment.

The coffee genome hub: a resource for coffee genomes

The whole genome sequence of Coffea canephora, the perennial diploid species known as Robusta, has been recently released. In the context of the C. canephora genome sequencing project and to support post-genomics efforts, we developed the Coffee Genome Hub (http://coffee-genome.org/), an integrative genome information system that allows centralized access to genomics and genetics data and analysis tools to facilitate translational and applied research in coffee. We provide the complete genome sequence of C. canephora along with gene structure, gene product information, metabolism, gene families, transcriptomics, syntenic blocks, genetic markers and genetic maps. The hub relies on generic software (e.g. GMOD tools) for easy querying, visualizing and downloading research data. It includes a Genome Browser enhanced by a Community Annotation System, enabling the improvement of automatic gene annotation through an annotation editor. In addition, the hub aims at developing interoperability among other existing South Green tools managing coffee data (phylogenomics resources, SNPs) and/or supporting data analyses with the Galaxy workflow manager.

Molecular characterization of an ethylene receptor gene (CcETR1) in coffee trees, its relationship with fruit development and caffeine content.

To understand the importance of ethylene receptor genes in the quality of coffee berries three full-length cDNAs corresponding to a putative ethylene receptor gene (ETR1) were isolated from Coffea canephora cDNA libraries. They differed by their 3′UTR and contained a main ORF and a 5′UTR short ORF putatively encoding a small polypeptide. The CcETR1 gene, present as a single copy in the C. canephora genome, contained five introns in the coding region and one in its 5′UTR. Alternative splicing can occur in C. canephora and C. pseudozanguebariae, leading to a truncated polypeptide. C. pseudozanguebariaeETR1 transcripts showed various forms of splicing alterations. This gene was equally expressed at all stages of fruit development. A segregation study on an inter-specific progeny showed that ETR1 is related to the fructification time, the caffeine content of the green beans, and seed weight. Arabidopsis transformed etiolated seedlings, which over-expressed CcETR1, displayed highly reduced gravitropism, but the triple response was observed in an ethylene enriched environment. These plants behaved like a low-concentration ethylene-insensitive mutant thus confirming the receptor function of the encoded protein. This gene showed no induction during the climacteric crisis but some linkage with traits related to quality.

Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae)

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1-copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution.

MoccaDB - an integrative database for functional, comparative and diversity studies in the Rubiaceae family.

BackgroundIn the past few years, functional genomics information has been rapidly accumulating on Rubiaceae species and especially on those belonging to the Coffea genus (coffee trees). An increasing number of expressed sequence tag (EST) data and EST- or genomic-derived microsatellite markers have been generated, together with Conserved Ortholog Set (COS) markers. This considerably facilitates comparative genomics or map-based genetic studies through the common use of orthologous loci across different species. Similar genomic information is available for e.g. tomato or potato, members of the Solanaceae family. Since both Rubiaceae and Solanaceae belong to the Euasterids I (lamiids) integration of information on genetic markers would be possible and lead to more efficient analyses and discovery of key loci involved in important traits such as fruit development, quality, and maturation, or adaptation. Our goal was to develop a comprehensive web data source for integrated information on validated orthologous markers in Rubiaceae.DescriptionMoccaDB is an online MySQL-PHP driven relational database that houses annotated and/or mapped microsatellite markers in Rubiaceae. In its current release, the database stores 638 markers that have been defined on 259 ESTs and 379 genomic sequences. Marker information was retrieved from 11 published works, and completed with original data on 132 microsatellite markers validated in our laboratory. DNA sequences were derived from three Coffea species/hybrids. Microsatellite markers were checked for similarity, in vitro tested for cross-amplification and diversity/polymorphism status in up to 38 Rubiaceae species belonging to the Cinchonoideae and Rubioideae subfamilies. Functional annotation was provided and some markers associated with described metabolic pathways were also integrated. Users can search the database for marker, sequence, map or diversity information through multi-option query forms. The retrieved data can be browsed and downloaded, along with protocols used, using a standard web browser. MoccaDB also integrates bioinformatics tools (CMap viewer and local BLAST) and hyperlinks to related external data sources (NCBI GenBank and PubMed, SOL Genomic Network database).ConclusionWe believe that MoccaDB will be extremely useful for all researchers working in the areas of comparative and functional genomics and molecular evolution, in general, and population analysis and association mapping of Rubiaceae and Solanaceae species, in particular.

Ancestral synteny shared between distantly-related plant species from the asterid (Coffea canephora and Solanum Sp.) and rosid (Vitis vinifera) clades

BackgroundCoffee trees (Rubiaceae) and tomato (Solanaceae) belong to the Asterid clade, while grapevine (Vitaceae) belongs to the Rosid clade. Coffee and tomato separated from grapevine 125 million years ago, while coffee and tomato diverged 83-89 million years ago. These long periods of divergent evolution should have permitted the genomes to reorganize significantly. So far, very few comparative mappings have been performed between very distantly related species belonging to different clades. We report the first multiple comparison between species from Asterid and Rosid clades, to examine both macro-and microsynteny relationships.ResultsThanks to a set of 867 COSII markers, macrosynteny was detected between coffee, tomato and grapevine. While coffee and tomato genomes share 318 orthologous markers and 27 conserved syntenic segments (CSSs), coffee and grapevine also share a similar number of syntenic markers and CSSs: 299 and 29 respectively. Despite large genome macrostructure reorganization, several large chromosome segments showed outstanding macrosynteny shedding new insights into chromosome evolution between Asterids and Rosids. We also analyzed a sequence of 174 kb containing the ovate gene, conserved in a syntenic block between coffee, tomato and grapevine that showed a high-level of microstructure conservation. A higher level of conservation was observed between coffee and grapevine, both woody and long life-cycle plants, than between coffee and tomato. Out of 16 coffee genes of this syntenic segment, 7 and 14 showed complete synteny between coffee and tomato or grapevine, respectively.ConclusionsThese results show that significant conservation is found between distantly related species from the Asterid (Coffea canephora and Solanum sp.) and Rosid (Vitis vinifera) clades, at the genome macrostructure and microstructure levels. At the ovate locus, conservation did not decline in relation to increasing phylogenetic distance, suggesting that the time factor alone does not explain divergences. Our results are considerably useful for syntenic studies between supposedly remote species for the isolation of important genes for agronomy.