Ecological and genomic vulnerability to climate change across native populations of Robusta coffee (Coffea canephora).

The assessment of population vulnerability under climate change is crucial for planning conservation as well as for ensuring food security. Coffea canephora is, in its native habitat, an understorey tree that is mainly distributed in the lowland rainforests of tropical Africa. Also known as Robusta, its commercial value constitutes a significant revenue for many human populations in tropical countries. Comparing ecological and genomic vulnerabilities within the species' native range can provide valuable insights about habitat loss and the species' adaptive potential, allowing to identify genotypes that may act as a resource for varietal improvement. By applying species distribution models, we assessed ecological vulnerability as the decrease in climatic suitability under future climatic conditions from 492 occurrences. We then quantified genomic vulnerability (or risk of maladaptation) as the allelic composition change required to keep pace with predicted climate change. Genomic vulnerability was estimated from genomic environmental correlations throughout the native range. Suitable habitat was predicted to diminish to half its size by 2050, with populations near coastlines and around the Congo River being the most vulnerable. Whole-genome sequencing revealed 165 candidate SNPs associated with climatic adaptation in C. canephora, which were located in genes involved in plant response to biotic and abiotic stressors. Genomic vulnerability was higher for populations in West Africa and in the region at the border between DRC and Uganda. Despite an overall low correlation between genomic and ecological vulnerability at broad scale, these two components of vulnerability overlap spatially in ways that may become damaging. Genomic vulnerability was estimated to be 23% higher in populations where habitat will be lost in 2050 compared to regions where habitat will remain suitable. These results highlight how ecological and genomic vulnerabilities are relevant when planning on how to cope with climate change regarding an economically important species.

Adaptive potential of Coffea canephora from Uganda in response to climate change.

Understanding vulnerabilities of plant populations to climate change could help preserve their biodiversity and reveal new elite parents for future breeding programmes. To this end, landscape genomics is a useful approach for assessing putative adaptations to future climatic conditions, especially in long-lived species such as trees. We conducted a population genomics study of 207 Coffea canephora trees from seven forests along different climate gradients in Uganda. For this, we sequenced 323 candidate genes involved in key metabolic and defence pathways in coffee. Seventy-one single nucleotide polymorphisms (SNPs) were found to be significantly associated with bioclimatic variables, and were thereby considered as putatively adaptive loci. These SNPs were linked to key candidate genes, including transcription factors, like DREB-like and MYB family genes controlling plant responses to abiotic stresses, as well as other genes of organoleptic interest, such as the DXMT gene involved in caffeine biosynthesis and a putative pest repellent. These climate-associated genetic markers were used to compute genetic offsets, predicting population responses to future climatic conditions based on local climate change forecasts. Using these measures of maladaptation to future conditions, substantial levels of genetic differentiation between present and future diversity were estimated for all populations and scenarios considered. The populations from the forests Zoka and Budongo, in the northernmost zone of Uganda, appeared to have the lowest genetic offsets under all predicted climate change patterns, while populations from Kalangala and Mabira, in the Lake Victoria region, exhibited the highest genetic offsets. The potential of these findings in terms of ex situ conservation strategies are discussed.

Genetic diversity of native and cultivated Ugandan Robusta coffee (Coffea canephora Pierre ex A. Froehner): Climate influences, breeding potential and diversity conservation.

Wild genetic resources and their ability to adapt to environmental change are critically important in light of the projected climate change, while constituting the foundation of agricultural sustainability. To address the expected negative effects of climate change on Robusta coffee trees (Coffea canephora), collecting missions were conducted to explore its current native distribution in Uganda over a broad climatic range. Wild material from seven forests could thus be collected. We used 19 microsatellite (SSR) markers to assess genetic diversity and structure of this material as well as material from two ex-situ collections and a feral population. The Ugandan C. canephora diversity was then positioned relative to the species' global diversity structure. Twenty-two climatic variables were used to explore variations in climatic zones across the sampled forests. Overall, Uganda's native C. canephora diversity differs from other known genetic groups of this species. In northwestern (NW) Uganda, four distinct genetic clusters were distinguished being from Zoka, Budongo, Itwara and Kibale forests A large southern-central (SC) cluster included Malabigambo, Mabira, and Kalangala forest accessions, as well as feral and cultivated accessions, suggesting similarity in genetic origin and strong gene flow between wild and cultivated compartments. We also confirmed the introduction of Congolese varieties into the SC region where most Robusta coffee production takes place. Identified populations occurred in divergent environmental conditions and 12 environmental variables significantly explained 16.3% of the total allelic variation across populations. The substantial genetic variation within and between Ugandan populations with different climatic envelopes might contain adaptive diversity to cope with climate change. The accessions that we collected have substantially enriched the diversity hosted in the Ugandan collections and thus contribute to ex situ conservation of this vital genetic resource. However, there is an urgent need to develop strategies to enhance complementary in-situ conservation of Coffea canephora in native forests in northwestern Uganda.

Development and evaluation of a genome-wide Coffee 8.5K SNP array and its application for high-density genetic mapping and for investigating the origin of Coffea arabica L.

Coffee species such as Coffea canephora P. (Robusta) and C. arabica L. (Arabica) are important cash crops in tropical regions around the world. C. arabica is an allotetraploid (2n = 4x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2n = 2x = 22). Interestingly, these progenitor species harbour a greater level of genetic variability and are an important source of genes to broaden the narrow Arabica genetic base. Here, we describe the development, evaluation and use of a single-nucleotide polymorphism (SNP) array for coffee trees. A total of 8580 unique and informative SNPs were selected from C. canephora and C. arabica sequencing data, with 40% of the SNP located in annotated genes. In particular, this array contains 227 markers associated to 149 genes and traits of agronomic importance. Among these, 7065 SNPs (~82.3%) were scorable and evenly distributed over the genome with a mean distance of 54.4 Kb between markers. With this array, we improved the Robusta high-density genetic map by adding 1307 SNP markers, whereas 945 SNPs were found segregating in the Arabica mapping progeny. A panel of C. canephora accessions was successfully discriminated and over 70% of the SNP markers were transferable across the three species. Furthermore, the canephora-derived subgenome of C. arabica was shown to be more closely related to C. canephora accessions from northern Uganda than to other current populations. These validated SNP markers and high-density genetic maps will be useful to molecular genetics and for innovative approaches in coffee breeding.

Structure and Distribution of Centromeric Retrotransposons at Diploid and Allotetraploid Coffea Centromeric and Pericentromeric Regions.

Centromeric regions of plants are generally composed of large array of satellites from a specific lineage of Gypsy LTR-retrotransposons, called Centromeric Retrotransposons. Repeated sequences interact with a specific H3 histone, playing a crucial function on kinetochore formation. To study the structure and composition of centromeric regions in the genus Coffea, we annotated and classified Centromeric Retrotransposons sequences from the allotetraploid C. arabica genome and its two diploid ancestors: Coffea canephora and C. eugenioides. Ten distinct CRC (Centromeric Retrotransposons in Coffea) families were found. The sequence mapping and FISH experiments of CRC Reverse Transcriptase domains in C. canephora, C. eugenioides, and C. arabica clearly indicate a strong and specific targeting mainly onto proximal chromosome regions, which can be associated also with heterochromatin. PacBio genome sequence analyses of putative centromeric regions on C. arabica and C. canephora chromosomes showed an exceptional density of one family of CRC elements, and the complete absence of satellite arrays, contrasting with usual structure of plant centromeres. Altogether, our data suggest a specific centromere organization in Coffea, contrasting with other plant genomes.

Two disjunct Pleistocene populations and anisotropic postglacial expansion shaped the current genetic structure of the relict plant Amborella trichopoda.

Past climate fluctuations shaped the population dynamics of organisms in space and time, and have impacted their present intra-specific genetic structure. Demo-genetic modelling allows inferring the way past demographic and migration dynamics have determined this structure. Amborella trichopoda is an emblematic relict plant endemic to New Caledonia, widely distributed in the understory of non-ultramafic rainforests. We assessed the influence of the last glacial climates on the demographic history and the paleo-distribution of 12 Amborella populations covering the whole current distribution. We performed coalescent genetic modelling of these dynamics, based on both whole-genome resequencing and microsatellite genotyping data. We found that the two main genetic groups of Amborella were shaped by the divergence of two ancestral populations during the last glacial maximum. From 12,800 years BP, the South ancestral population has expanded 6.3-fold while the size of the North population has remained stable. Recent asymmetric gene flow between the groups further contributed to the phylogeographical pattern. Spatially explicit coalescent modelling allowed us to estimate the location of ancestral populations with good accuracy (< 22 km) and provided indications regarding the mid-elevation pathways that facilitated post-glacial expansion.

Distribution of Divo in Coffea genomes, a poorly described family of angiosperm LTR-Retrotransposons.

Coffea arabica (the Arabica coffee) is an allotetraploid species originating from a recent hybridization between two diploid species: C. canephora and C. eugenioides. Transposable elements can drive structural and functional variation during the process of hybridization and allopolyploid formation in plants. To learn more about the evolution of the C. arabica genome, we characterized and studied a new Copia LTR-Retrotransposon (LTR-RT) family in diploid and allotetraploid Coffea genomes called Divo. It is a complete and relatively compact LTR-RT element (~5 kb), carrying typical Gag and Pol Copia type domains. Reverse Trancriptase (RT) domain-based phylogeny demonstrated that Divo is a new and well-supported family in the Bianca lineage, but strictly restricted to dicotyledonous species. In C. canephora, Divo is expressed and showed a genomic distribution along gene rich and gene poor regions. The copy number, the molecular estimation of insertion time and the analysis at orthologous locations of insertions in diploid and allotetraploid coffee genomes suggest that Divo underwent a different and recent transposition activity in C. arabica and C. canephora when compared to C. eugenioides. The analysis of this novel LTR-RT family represents an important step toward uncovering the genome structure and evolution of C. arabica allotetraploid genome.

Genotyping-by-sequencing provides the first well-resolved phylogeny for coffee (Coffea) and insights into the evolution of caffeine content in its species: GBS coffee phylogeny and the evolution of caffeine content.

A comprehensive and meaningful phylogenetic hypothesis for the commercially important coffee genus (Coffea) has long been a key objective for coffee researchers. For molecular studies, progress has been limited by low levels of sequence divergence, leading to insufficient topological resolution and statistical support in phylogenetic trees, particularly for the major lineages and for the numerous species occurring in Madagascar. We report here the first almost fully resolved, broadly sampled phylogenetic hypothesis for coffee, the result of combining genotyping-by-sequencing (GBS) technology with a newly developed, lab-based workflow to integrate short read next-generation sequencing for low numbers of additional samples. Biogeographic patterns indicate either Africa or Asia (or possibly the Arabian Peninsula) as the most likely ancestral locality for the origin of the coffee genus, with independent radiations across Africa, Asia, and the Western Indian Ocean Islands (including Madagascar and Mauritius). The evolution of caffeine, an important trait for commerce and society, was evaluated in light of our phylogeny. High and consistent caffeine content is found only in species from the equatorial, fully humid environments of West and Central Africa, possibly as an adaptive response to increased levels of pest predation. Moderate caffeine production, however, evolved at least one additional time recently (between 2 and 4Mya) in a Madagascan lineage, which suggests that either the biosynthetic pathway was already in place during the early evolutionary history of coffee, or that caffeine synthesis within the genus is subject to convergent evolution, as is also the case for caffeine synthesis in coffee versus tea and chocolate.

Partial sequencing reveals the transposable element composition of Coffea genomes and provides evidence for distinct evolutionary stories.

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.

Shift in precipitation regime promotes interspecific hybridization of introduced Coffea species.

The frequency of plant species introductions has increased in a highly connected world, modifying species distribution patterns to include areas outside their natural ranges. These introductions provide the opportunity to gain new insight into the importance of flowering phenology as a component of adaptation to a new environment. Three Coffea species, C. arabica, C. canephora (Robusta), and C. liberica, native to intertropical Africa have been introduced to New Caledonia. On this archipelago, a secondary contact zone has been characterized where these species coexist, persist, and hybridize spontaneously. We investigated the impact of environmental changes undergone by each species following its introduction in New Caledonia on flowering phenology and overcoming reproductive barriers between sister species. We developed species distribution models and compared both environmental envelopes and climatic niches between native and introduced hybrid zones. Flowering phenology was monitored in a population in the hybrid zone along with temperature and precipitation sequences recorded at a nearby weather station. The extent and nature of hybridization events were characterized using chloroplast and nuclear microsatellite markers. The three Coffea species encountered weak environmental suitability compared to their native ranges when introduced to New Caledonia, especially C. arabica and C. canephora. The niche of the New Caledonia hybrid zone was significantly different from all three species' native niches based on identity tests (I Similarity and D Schoener's Similarity Indexes). This area appeared to exhibit intermediate conditions between the native conditions of the three species for temperature-related variables and divergent conditions for precipitation-related ones. Flowering pattern in these Coffea species was shown to have a strong genetic component that determined the time between the triggering rain and anthesis (flower opening), specific to each species. However, a precipitation regime different from those in Africa was directly involved in generating partial flowering overlap between species and thus in allowing hybridization and interspecific gene flow. Interspecific hybrids accounted for 4% of the mature individuals in the sympatric population and occurred between each pair of species with various level of introgression. Adaptation to new environmental conditions following introduction of Coffea species to New Caledonia has resulted in a secondary contact between three related species, which would not have happened in their native ranges, leading to hybridization and gene flow.

Active transposable elements recover species boundaries and geographic structure in Madagascan coffee species.

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.

The Amborella vacuolar processing enzyme family.

Most vacuolar proteins are synthesized on rough endoplasmic reticulum as proprotein precursors and then transported to the vacuoles, where they are converted into their respective mature forms by vacuolar processing enzymes (VPEs). In the case of the seed storage proteins, this process is of major importance, as it conditions the establishment of vigorous seedlings. Toward the goal of identifying proteome signatures that could be associated with the origin and early diversification of angiosperms, we previously characterized the 11S-legumin-type seed storage proteins from Amborella trichopoda, a rainforest shrub endemic to New Caledonia that is also the probable sister to all other angiosperms (Amborella Genome Project, 2013). In the present study, proteomic and genomic approaches were used to characterize the VPE family in this species. Three genes were found to encode VPEs in the Amborella's genome. Phylogenetic analyses showed that the Amborella sequences grouped within two major clades of angiosperm VPEs, indicating that the duplication that generated the ancestors of these clades occurred before the most recent common ancestor of living angiosperms. A further important duplication within the VPE family appears to have occurred in common ancestor of the core eudicots, while many more recent duplications have also occurred in specific taxa, including both Arabidopsis thaliana and Amborella. An analysis of natural genetic variation for each of the three Amborella VPE genes revealed the absence of selective forces acting on intronic and exonic single-nucleotide polymorphisms among several natural Amborella populations in New Caledonia.

Large distribution and high sequence identity of a Copia-type retrotransposon in angiosperm families.

Retrotransposons are the main component of plant genomes. Recent studies have revealed the complexity of their evolutionary dynamics. Here, we have identified Copia25 in Coffea canephora, a new plant retrotransposon belonging to the Ty1-Copia superfamily. In the Coffea genomes analyzed, Copia25 is present in relatively low copy numbers and transcribed. Similarity sequence searches and PCR analyses show that this retrotransposon with LTRs (Long Terminal Repeats) is widely distributed among the Rubiaceae family and that it is also present in other distantly related species belonging to Asterids, Rosids and monocots. A particular situation is the high sequence identity found between the Copia25 sequences of Musa, a monocot, and Ixora, a dicot species (Rubiaceae). Our results reveal the complexity of the evolutionary dynamics of the ancient element Copia25 in angiosperm, involving several processes including sequence conservation, rapid turnover, stochastic losses and horizontal transfer.

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.

BAC-end sequences analysis provides first insights into coffee (Coffea canephora P.) genome composition and evolution.

Coffee is one of the world's most important agricultural commodities. Coffee belongs to the Rubiaceae family in the euasterid I clade of dicotyledonous plants, to which the Solanaceae family also belongs. Two bacterial artificial chromosome (BAC) libraries of a homozygous doubled haploid plant of Coffea canephora were constructed using two enzymes, HindIII and BstYI. A total of 134,827 high quality BAC-end sequences (BESs) were generated from the 73,728 clones of the two libraries, and 131,412 BESs were conserved for further analysis after elimination of chloroplast and mitochondrial sequences. This corresponded to almost 13 % of the estimated size of the C. canephora genome. 6.7 % of BESs contained simple sequence repeats, the most abundant (47.8 %) being mononucleotide motifs. These sequences allow the development of numerous useful marker sites. Potential transposable elements (TEs) represented 11.9 % of the full length BESs. A difference was observed between the BstYI and HindIII libraries (14.9 vs. 8.8 %). Analysis of BESs against known coding sequences of TEs indicated that 11.9 % of the genome corresponded to known repeat sequences, like for other flowering plants. The number of genes in the coffee genome was estimated at 41,973 which is probably overestimated. Comparative genome mapping revealed that microsynteny was higher between coffee and grapevine than between coffee and tomato or Arabidopsis. BESs constitute valuable resources for the first genome wide survey of coffee and provide new insights into the composition and evolution of the coffee genome.

Genetic structure and diversity of coffee (Coffea) across Africa and the Indian Ocean islands revealed using microsatellites.

BACKGROUND AND AIMS: The 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. METHODS: Genetic 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 RESULTS: Across 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. CONCLUSIONS: Cross-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.

Microsatellite markers for Amborella (Amborellaceae), a monotypic genus endemic to New Caledonia.

PREMISE OF THE STUDY: Informative markers are required for assessing the diversity of Amborella trichopoda, the only species of its order, endemic to New Caledonia and considered to be the sister species to all flowering plants. Therefore, expressed sequence tag (EST)-based microsatellite markers were developed. • METHODS AND RESULTS: Fifty-five microsatellite loci were characterized in 14896 putative unigenes, which were generated by assembling A. trichopoda ESTs from the public sequence database. Seventeen markers revealed polymorphism in 80 adult shrubs from three populations. The number of alleles per locus ranged from two to 12, with a total of 132 alleles scored. The mean expected heterozygosity per population ranged from 0.336 to 0.567. • CONCLUSIONS: These markers offer an appropriate amount of variation to investigate genetic diversity structure, gene flow, and other conservation issues.

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

BACKGROUND: Coffee 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. RESULTS: Thanks 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. CONCLUSIONS: These 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.

Characterization, high-resolution mapping and differential expression of three homologous PAL genes in Coffea canephora Pierre (Rubiaceae).

Phenylalanine ammonia lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway producing phenolics, widespread constituents of plant foods and beverages, including chlorogenic acids, polyphenols found at remarkably high levels in the coffee bean and long recognized as powerful antioxidants. To date, whereas PAL is generally encoded by a small gene family, only one gene has been characterized in Coffea canephora (CcPAL1), an economically important species of cultivated coffee. In this study, a molecular- and bioinformatic-based search for CcPAL1 paralogues resulted successfully in identifying two additional genes, CcPAL2 and CcPAL3, presenting similar genomic structures and encoding proteins with close sequences. Genetic mapping helped position each gene in three different coffee linkage groups, CcPAL2 in particular, located in a coffee genome linkage group (F) which is syntenic to a region of Tomato Chromosome 9 containing a PAL gene. These results, combined with a phylogenetic study, strongly suggest that CcPAL2 may be the ancestral gene of C. canephora. A quantitative gene expression analysis was also conducted in coffee tissues, showing that all genes are transcriptionally active, but they present distinct expression levels and patterns. We discovered that CcPAL2 transcripts appeared predominantly in flower, fruit pericarp and vegetative/lignifying tissues like roots and branches, whereas CcPAL1 and CcPAL3 were highly expressed in immature fruit. This is the first comprehensive study dedicated to PAL gene family characterization in coffee, allowing us to advance functional studies which are indispensable to learning to decipher what role this family plays in channeling the metabolism of coffee phenylpropanoids.

Site-Specific Insertion Polymorphism of the MITE Alex-1 in the Genus Coffea Suggests Interspecific Gene Flow.

Miniature Inverted-repeat Transposable Elements (MITEs) are small nonautonomous class-II transposable elements distributed throughout eukaryotic genomes. We identified a novel family of MITEs (named Alex) in the Coffea canephora genome often associated with expressed sequences. The Alex-1 element is inserted in an intron of a gene at the CcEIN4 locus. Its mobility was demonstrated by sequencing the insertion site in C. canephora accessions and Coffea species. Analysis of the insertion polymorphism of Alex-1 at this locus in Coffea species and in C. canephora showed that there was no relationship between the geographical distribution of the species, their phylogenetic relationships, and insertion polymorphism. The intraspecific distribution of C. canephora revealed an original situation within the E diversity group. These results suggest possibly greater gene flow between species than previously thought. This MITE family will enable the study of the C. canephora genome evolution, phylogenetic relationships, and possible gene flows within the Coffea genus.