New cup out of old coffee: contribution of parental gene expression legacy to phenotypic novelty in coffee beans of the allopolyploid Coffea arabica L.

BACKGROUND AND AIMS: Allopolyploidization is a widespread phenomenon known to generate novel phenotypes by merging evolutionarily distinct parental genomes and regulatory networks in a single nucleus. The objective of this study was to investigate the transcriptional regulation associated with phenotypic novelty in coffee beans of the allotetraploid Coffea arabica. METHODS: A genome-wide comparative transcriptomic analysis was performed in C. arabica and its two diploid progenitors, C. canephora and C. eugenioides. Gene expression patterns and homeologue expression were studied on seeds at five different maturation stages. The involvement of homeologue expression bias (HEB) in specific traits was addressed both by functional enrichment analyses and by the study of gene expression in the caffeine and chlorogenic acid biosynthesis pathways. KEY RESULTS: Expression-level dominance in C. arabica seed was observed for most of the genes differentially expressed between the species. Approximately a third of the genes analysed showed HEB. This proportion increased during seed maturation but the biases remained equally distributed between the sub-genomes. The relative expression levels of homeologues remained relatively constant during maturation and were correlated with those estimated in leaves of C. arabica and interspecific hybrids between C. canephora and C. eugenioides. Functional enrichment analyses performed on genes exhibiting HEB enabled the identification of processes potentially associated with physiological traits. The expression profiles of the genes involved in caffeine biosynthesis mirror the differences observed in the caffeine content of mature seeds of C. arabica and its parental species. CONCLUSIONS: Neither of the two sub-genomes is globally preferentially expressed in C. arabica seeds, and homeologues appear to be co-regulated by shared trans-regulatory mechanisms. The observed HEBs are thought to be a legacy of gene expression differences inherited from diploid progenitor species. Pre-existing functional divergences between parental species appear to play an important role in controlling the phenotype of C. arabica seeds.

Multi-Approach Analysis Reveals Local Adaptation in a Widespread Forest Tree of Reunion Island.

Detecting processes of local adaptation in forest trees and identifying environmental selective drivers are of primary importance for forest management and conservation. Transplant experiments, functional genomics and population genomics are complementary tools to efficiently characterize heritable phenotypic traits and to decipher the genetic bases of adaptive traits. Using an integrative approach combining phenotypic assessment in common garden, transcriptomics and landscape genomics, we investigated leaf adaptive traits in Coffea mauritiana, a forest tree endemic to Reunion Island. Eight populations of C. mauritiana originating from sites with contrasted environmental conditions were sampled in common garden to assess several leaf morphological traits, to analyze the leaf transcriptome and leaf cuticular wax composition. The relative alkane content of cuticular waxes was significantly correlated with major climatic gradients, paving the way for further transcriptome-based analyses. The expression pattern of cuticle biosynthetic genes was consistent with a modulation of alkane accumulation across the population studied, supporting the hypothesis that the composition of cuticular wax is involved in the local adaptation of C. mauritiana. Association tests in landscape genomics performed using RNA-seq-derived single-nucleotide polymorphisms revealed that genes associated with cell wall remodeling also likely play an adaptive role. By combining these different approaches, this study efficiently identified local adaptation processes in a non-model species. Our results provide the first evidence for local adaptation in trees endemic to Reunion Island and highlight the importance of cuticle composition for the adaptation of trees to the high evaporative demand in warm climates.

Seed comparative genomics in three coffee species identify desiccation tolerance mechanisms in intermediate seeds.

In contrast to desiccation-tolerant 'orthodox' seeds, so-called 'intermediate' seeds cannot survive complete drying and are short-lived. All species of the genus Coffea produce intermediate seeds, but they show a considerable variability in seed desiccation tolerance (DT), which may help to decipher the molecular basis of seed DT in plants. We performed a comparative transcriptome analysis of developing seeds in three coffee species with contrasting desiccation tolerance. Seeds of all species shared a major transcriptional switch during late maturation that governs a general slow-down of metabolism. However, numerous key stress-related genes, including those coding for the late embryogenesis abundant protein EM6 and the osmosensitive calcium channel ERD4, were up-regulated during DT acquisition in the two species with high seed DT, C. arabica and C. eugenioides. By contrast, we detected up-regulation of numerous genes involved in the metabolism, transport, and perception of auxin in C. canephora seeds with low DT. Moreover, species with high DT showed a stronger down-regulation of the mitochondrial machinery dedicated to the tricarboxylic acid cycle and oxidative phosphorylation. Accordingly, respiration measurements during seed dehydration demonstrated that intermediate seeds with the highest DT are better prepared to cease respiration and avoid oxidative stresses.

Plant population dynamics on oceanic islands during the Late Quaternary climate changes: genetic evidence from a tree species (Coffea mauritiana) in Reunion Island.

Past climatic fluctuations have played a major role in shaping the current plant biodiversity. Although harbouring an exceptional biota, oceanic islands have received little attention in studies on species demographic history and past vegetation patterns. We investigated the impact of past climatic changes on the effective population size of a tree (Coffea mauritiana) that is endemic to Reunion Island, located in the south-western Indian Ocean (SWIO). Demographic changes were inferred using summary statistics calculated from genomic data. Using ecological niche modelling and the current distribution of genetic diversity, the paleodistribution of the species was also assessed. A reduction in the effective population size of C. mauritiana during the last glaciation maximum was inferred. The distribution of the species was reduced on the western side of the island, due to low rainfall. It appeared that a major reduction in rainfall and a slight temperature decrease prevailed in the SWIO. Our findings indicated that analyses on the current patterns of intraspecific genetic variations can efficiently contribute to past climatic changes characterisation in remote islands. Identifying area with higher resilience in oceanic islands could provide guidance in forest management and conservation faced to the global climate change.

Genetic diversity and population divergences of an indigenous tree (Coffea mauritiana) in Reunion Island: role of climatic and geographical factors.

Oceanic islands are commonly considered as natural laboratories for studies on evolution and speciation. The evolutionary specificities of islands associated with species biology provide unique scenarios to study the role of geography and climate in driving population divergence. However, few studies have addressed this subject in small oceanic islands with heterogeneous climates. Being widely distributed in Reunion Island forest, Coffea mauritiana represents an interesting model case for investigating patterns of within-island differentiation at small spatial scale. In this study, we examined the genetic diversity and population divergences of C. mauritiana using SNP markers obtained from 323 individuals across 34 locations in Reunion Island. Using redundancy analysis, we further evaluated the contribution of geographic and climatic factors to shaping genetic divergence among populations. Genetic diversity analyses revealed that accessions clustered according to the source population, with further grouping in regional clusters. Genetic relationships among the regional clusters underlined a recent process of expansion in the form of step-by-step colonization on both sides of the island. Divergence among source populations was mostly driven by the joint effect of geographic distance and climatic heterogeneity. The pattern of isolation-by-geography was in accordance with the dispersal characteristics of the species, while isolation-by-environment was mostly explained by the heterogeneous rainfall patterns, probably associated with an asynchronous flowering among populations. These findings advance our knowledge on the patterns of genetic diversity and factors of population differentiation of species native to Reunion Island, and will also usefully guide forest management for conservation.

Integrative analysis of the late maturation programme and desiccation tolerance mechanisms in intermediate coffee seeds.

The 'intermediate seed' category was defined in the early 1990s using coffee (Coffea arabica) as a model. In contrast to orthodox seeds, intermediate seeds cannot survive complete drying, which is a major constraint for seed storage and has implications for both biodiversity conservation and agricultural purposes. However, intermediate seeds are considerably more tolerant to drying than recalcitrant seeds, which are highly sensitive to desiccation. To gain insight into the mechanisms governing such differences, changes in desiccation tolerance (DT), hormone contents, and the transcriptome were analysed in developing coffee seeds. Acquisition of DT coincided with a dramatic transcriptional switch characterised by the repression of primary metabolism, photosynthesis, and respiration, and the up-regulation of genes coding for late-embryogenesis abundant (LEA) proteins, heat-shock proteins (HSPs), and antioxidant enzymes. Analysis of the heat-stable proteome in mature coffee seeds confirmed the accumulation of LEA proteins identified at the transcript level. Transcriptome analysis also suggested a major role for ABA and for the transcription factors CaHSFA9, CaDREB2G, CaANAC029, CaPLATZ, and CaDOG-like in DT acquisition. The ability of CaHSFA9 and CaDREB2G to trigger HSP gene transcription was validated by Agrobacterium-mediated transformation of coffee somatic embryos.

Inter-genomic DNA Exchanges and Homeologous Gene Silencing Shaped the Nascent Allopolyploid Coffee Genome (Coffea arabica L.).

Allopolyploidization is a biological process that has played a major role in plant speciation and evolution. Genomic changes are common consequences of polyploidization, but their dynamics over time are still poorly understood. Coffea arabica, a recently formed allotetraploid, was chosen to study genetic changes that accompany allopolyploid formation. Both RNA-seq and DNA-seq data were generated from two genetically distant C. arabica accessions. Genomic structural variation was investigated using C. canephora, one of its diploid progenitors, as reference genome. The fate of 9047 duplicate homeologous genes was inferred and compared between the accessions. The pattern of SNP density along the reference genome was consistent with the allopolyploid structure. Large genomic duplications or deletions were not detected. Two homeologous copies were retained and expressed in 96% of the genes analyzed. Nevertheless, duplicated genes were found to be affected by various genomic changes leading to homeolog loss or silencing. Genetic and epigenetic changes were evidenced that could have played a major role in the stabilization of the unique ancestral allotetraploid and its subsequent diversification. While the early evolution of C. arabica mainly involved homeologous crossover exchanges, the later stage appears to have relied on more gradual evolution involving gene conversion and homeolog silencing.

Gene coexpression network analysis of oil biosynthesis in an interspecific backcross of oil palm.

Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for ß-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops.

Regulatory divergence between parental alleles determines gene expression patterns in hybrids.

Both hybridization and allopolyploidization generate novel phenotypes by conciliating divergent genomes and regulatory networks in the same cellular context. To understand the rewiring of gene expression in hybrids, the total expression of 21,025 genes and the allele-specific expression of over 11,000 genes were quantified in interspecific hybrids and their parental species, Coffea canephora and Coffea eugenioides using RNA-seq technology. Between parental species, cis- and trans-regulatory divergences affected around 32% and 35% of analyzed genes, respectively, with nearly 17% of them showing both. The relative importance of trans-regulatory divergences between both species could be related to their low genetic divergence and perennial habit. In hybrids, among divergently expressed genes between parental species and hybrids, 77% was expressed like one parent (expression level dominance), including 65% like C. eugenioides. Gene expression was shown to result from the expression of both alleles affected by intertwined parental trans-regulatory factors. A strong impact of C. eugenioides trans-regulatory factors on the upregulation of C. canephora alleles was revealed. The gene expression patterns appeared determined by complex combinations of cis- and trans-regulatory divergences. In particular, the observed biased expression level dominance seemed to be derived from the asymmetric effects of trans-regulatory parental factors on regulation of alleles. More generally, this study illustrates the effects of divergent trans-regulatory parental factors on the gene expression pattern in hybrids. The characteristics of the transcriptional response to hybridization appear to be determined by the compatibility of gene regulatory networks and therefore depend on genetic divergences between the parental species and their evolutionary history.

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.

Genome rearrangements derived from homoeologous recombination following allopolyploidy speciation in coffee.

Allopolyploidization is widespread and has played a major role in flowering plant diversification. Genomic changes are common consequences of allopolyploidization, but their mechanisms of occurrence and dynamics over time are still poorly understood. Coffea arabica, a recently formed allotetraploid, was chosen as a model to investigate genetic changes in allopolyploid using an approach that exploits next-generation sequencing technologies. Genes affected by putative homoeolog loss were inferred by comparing the numbers of single-nucleotide polymorphisms detected using RNA-seq in individual accessions of C. arabica, and between accessions of its two diploid progenitor species for common sequence positions. Their physical locations were investigated and clusters of genes exhibiting homoeolog loss were identified. To validate these results, genome sequencing data were generated from one accession of C. arabica and further analyzed. Genomic rearrangements involving homoeologous exchanges appear to occur in C. arabica and to be a major source of genetic diversity. At least 5% of the C. arabica genes were inferred to have undergone homoeolog loss. The detection of a large number of homoeologous exchange events (HEEs) shared by all accessions of C. arabica strongly reinforces the assumption of a single allopolyploidization event. Furthermore, HEEs were specific to one or a few accessions, suggesting that HEE accumulates gradually. Our results provide evidence for the important role of HEE in allopolyploid genome evolution.

Regulation of galactomannan biosynthesis in coffee seeds.

The seed of Coffea arabica accumulates large amounts of cell wall storage polysaccharides (CWSPs) of the mannan family in the cell walls of the endosperm. The variability induced by the growing environment and extensive pairwise correlation analysis with stringent significance thresholds was used to investigate transcript-transcript and transcript-metabolite relationships among 26 sugar-related genes, and the amount of CWSPs and seven soluble low molecular weight carbohydrates in the developing coffee endosperm. A dense module of nine quantitatively co-expressed genes was detected at the mid-developmental stage when CWSPs accumulate. This module included the five genes of the core galactomannan synthetic machinery, namely genes coding for the enzymes needed to assemble the mannan backbone (mannan synthase, ManS), and genes that introduce the galactosyl side chains (galactosyltransferase, GMGT), modulate the post-depositional degree of galactose substitution (α-galactosidase), and produce the nucleotide sugar building blocks GDP-mannose and UDP-galactose (mannose-1P guanyltransferase and UDP-glucose 4'-epimerase, respectively). The amount of CWSPs stored in the endosperm at the onset of their accumulation was primarily and quantitatively modulated at the transcriptional level (i.e. positively correlated with the expression level of these key galactomannan biosynthetic genes). This analysis also suggests a role for sorbitol and raffinose family oligosaccharides as transient auxiliary sources of building blocks for galactomannan synthesis. Finally, a microarray-based analysis of the developing seed transcriptome revealed that all genes of the core galactomannan synthesis machinery grouped in a single cluster of 209 co-expressed genes. Analysis of the gene composition of this cluster revealed remarkable functional coherence and identified transcription factors that putatively control galactomannan biosynthesis in coffee.

SNiPloid: A Utility to Exploit High-Throughput SNP Data Derived from RNA-Seq in Allopolyploid Species.

High-throughput sequencing is a common approach to discover SNP variants, especially in plant species. However, methods to analyze predicted SNPs are often optimized for diploid plant species whereas many crop species are allopolyploids and combine related but divergent subgenomes (homoeologous chromosome sets). We created a software tool, SNiPloid, that exploits and interprets putative SNPs in the context of allopolyploidy by comparing SNPs from an allopolyploid with those obtained in its modern-day diploid progenitors. SNiPloid can compare SNPs obtained from a sample to estimate the subgenome contribution to the transcriptome or SNPs obtained from two polyploid accessions to search for SNP divergence.

Contribution of subgenomes to the transcriptome and their intertwined regulation in the allopolyploid Coffea arabica grown at contrasted temperatures.

Polyploidy has occurred throughout the evolutionary history of plants and led to diversification and plant ecological adaptation. Functional plasticity of duplicate genes is believed to play a major role in the environmental adaptation of polyploids. In this context, we characterized genome-wide homoeologous gene expression in Coffea arabica, a recent allopolyploid combining two subgenomes that derive from two closely related diploid species, and investigated its variation in response to changing environment. The transcriptome of leaves of C. arabica cultivated at different growing temperatures suitable for one or the other parental species was examined using RNA-sequencing. The relative contribution of homoeologs to gene expression was estimated for 9959 and 10,628 genes in warm and cold conditions, respectively. Whatever the growing conditions, 65% of the genes showed equivalent levels of homoeologous gene expression. In 92% of the genes, relative homoeologous gene expression varied < 10% between growing temperatures. The subgenome contributions to the transcriptome appeared to be only marginally altered by the different conditions (involving intertwined regulations of homeologs) suggesting that C. arabica's ability to tolerate a broader range of growing temperatures than its diploid parents does not result from differential use of homoeologs.

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.

Differences in evolution rates among eudicotyledon species observed by analysis of protein divergence.

Genome evolution rates can vary considerably among plants. In particular, a correlation has often been reported between the evolution rate and annual/perennial habit, possibly associated with differences in generation time. For example, among the rosid species whose genome is fully sequenced, Vitis vinifera, a perennial species, was shown to have the genome that evolved the slowest. In order to extend knowledge of evolution rates to the asterid clade, one of the two major clades of core eudicotyledonous, the protein evolution rates in three asterid species, one perennial (Coffea canephora) and two annual species (Solanum lycopersicum and Mimulus guttatus), were investigated and compared with V. vinifera. Significant differences were observed among these species, and the proteins that evolved the most slowly were those of V. vinifera. Among the species belonging to the asterid clade, C. canephora appears to have evolved more slowly than the others. These findings are consistent with a correlation between perennial habit and slow evolution rates. The C. canephora genome seems to be an appropriate model for paleogenomic studies of asterids.

High genetic and epigenetic stability in Coffea arabica plants derived from embryogenic suspensions and secondary embryogenesis as revealed by AFLP, MSAP and the phenotypic variation rate.

Embryogenic suspensions that involve extensive cell division are risky in respect to genome and epigenome instability. Elevated frequencies of somaclonal variation in embryogenic suspension-derived plants were reported in many species, including coffee. This problem could be overcome by using culture conditions that allow moderate cell proliferation. In view of true-to-type large-scale propagation of C. arabica hybrids, suspension protocols based on low 2,4-D concentrations and short proliferation periods were developed. As mechanisms leading to somaclonal variation are often complex, the phenotypic, genetic and epigenetic changes were jointly assessed so as to accurately evaluate the conformity of suspension-derived plants. The effects of embryogenic suspensions and secondary embryogenesis, used as proliferation systems, on the genetic conformity of somatic embryogenesis-derived plants (emblings) were assessed in two hybrids. When applied over a 6 month period, both systems ensured very low somaclonal variation rates, as observed through massive phenotypic observations in field plots (0.74% from 200,000 plant). Molecular AFLP and MSAP analyses performed on 145 three year-old emblings showed that polymorphism between mother plants and emblings was extremely low, i.e. ranges of 0-0.003% and 0.07-0.18% respectively, with no significant difference between the proliferation systems for the two hybrids. No embling was found to cumulate more than three methylation polymorphisms. No relation was established between the variant phenotype (27 variants studied) and a particular MSAP pattern. Chromosome counting showed that 7 of the 11 variant emblings analyzed were characterized by the loss of 1-3 chromosomes. This work showed that both embryogenic suspensions and secondary embryogenesis are reliable for true-to-type propagation of elite material. Molecular analyses revealed that genetic and epigenetic alterations are particularly limited during coffee somatic embryogenesis. The main change in most of the rare phenotypic variants was aneuploidy, indicating that mitotic aberrations play a major role in somaclonal variation in coffee.

Genome evolution in diploid and tetraploid Coffea species as revealed by comparative analysis of orthologous genome segments.

Sequence comparison of orthologous regions enables estimation of the divergence between genomes, analysis of their evolution and detection of particular features of the genomes, such as sequence rearrangements and transposable elements. Despite the economic importance of Coffea species, little genomic information is currently available. Coffea is a relatively young genus that includes more than one hundred diploid species and a single tetraploid species. Three Coffea orthologous regions of 470-900 kb were analyzed and compared: both subgenomes of allotetraploid Coffea arabica (contributed by the diploid species Coffea eugenioides and Coffea canephora) and the genome of diploid C. canephora. Sequence divergence was calculated on global alignments or on coding and non-coding sequences separately. A search for transposable elements detected 43 retrotransposons and 198 transposons in the sequences analyzed. Comparative insertion analysis made it possible to locate 165 TE insertions in the phylogenetic tree of the three genomes/subgenomes. In the tetraploid C. arabica, a homoeologous non-reciprocal transposition (HNRT) was detected and characterized: a 50 kb region of the C. eugenioides derived subgenome replaced the C. canephora derived counterpart. Comparative sequence analysis on three Coffea genomes/subgenomes revealed almost perfect gene synteny, low sequence divergence and a high number of shared transposable elements. Compared to the results of similar analysis in other genera (Aegilops/Triticum and Oryza), Coffea genomes/subgenomes appeared to be dramatically less diverged, which is consistent with the relatively recent radiation of the Coffea genus. Based on nucleotide substitution frequency, the HNRT was dated at 10,000-50,000 years BP, which is also the most recent estimation of the origin of C. arabica.

Homeologous gene expression in response to growing temperature in a recent Allopolyploid (Coffea arabica L.).

Allopolyploidy is considered as a major factor contributing to speciation, diversification, and plant ecological adaptation. In particular, the expression of duplicate genes (homeologs) can be altered leading to functional plasticity and to phenotypic novelty. This study investigated the influence of growing temperatures on homeologous gene expression in Coffea arabica L., a recent allopolyploid involving 2 closely related diploid parental species. The relative expression of homeologs of 13 genes all located in the same genomic region was analyzed using an SNP ratio quantification method based on dideoxy-terminated sequences of cDNA amplicons. The relative expression of homeologous genes varied depending on the gene, the organ, and the growing condition. Nevertheless, expression of both homeologs was always detected (i.e., no silencing). Although the growing conditions were suitable for one or other of the parental species, neither subgenome appeared preferentially expressed. Furthermore, relative homeologous expression showed moderate variations across organs and conditions and appeared uncorrelated between adjacent genes. These results indicate the absence of signs of subfunctionalization suggesting C. arabica has not undergone noticeable diploidization. Furthermore, these results suggest that the expression of homeologous genes in C. arabica is regulated by a shared trans-regulation mechanism acting similarly on the 2 subgenomes and that the observed biases in the relative homeolog expression may result from cis fine-scale factors.