The genome and population genomics of allopolyploid Coffea arabica reveal the diversification history of modern coffee cultivars.

Coffea arabica, an allotetraploid hybrid of Coffea eugenioides and Coffea canephora, is the source of approximately 60% of coffee products worldwide, and its cultivated accessions have undergone several population bottlenecks. We present chromosome-level assemblies of a di-haploid C. arabica accession and modern representatives of its diploid progenitors, C. eugenioides and C. canephora. The three species exhibit largely conserved genome structures between diploid parents and descendant subgenomes, with no obvious global subgenome dominance. We find evidence for a founding polyploidy event 350,000-610,000 years ago, followed by several pre-domestication bottlenecks, resulting in narrow genetic variation. A split between wild accessions and cultivar progenitors occurred ~30.5 thousand years ago, followed by a period of migration between the two populations. Analysis of modern varieties, including lines historically introgressed with C. canephora, highlights their breeding histories and loci that may contribute to pathogen resistance, laying the groundwork for future genomics-based breeding of C. arabica.

Global transcriptome profiling reveals differential regulatory, metabolic and hormonal networks during somatic embryogenesis in Coffea arabica.

BACKGROUND: Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial and error approach. We report the first global scale transcriptome profiling performed at all developmental stages of SE in coffee to unravel the mechanisms that regulate cell fate and totipotency. RESULTS: RNA-seq of 48 samples (12 developmental stages × 4 biological replicates) generated 90 million high quality reads per sample, approximately 74% of which were uniquely mapped to the Arabica genome. First, the statistical analysis of transcript data clearly grouped SE developmental stages into seven important phases (Leaf, Dedifferentiation, Primary callus, Embryogenic callus, Embryogenic cell clusters, Redifferentiation and Embryo) enabling the identification of six key developmental phase switches, which are strategic for the overall biological efficiency of embryo regeneration. Differential gene expression and functional analysis showed that genes encoding transcription factors, stress-related genes, metabolism-related genes and hormone signaling-related genes were significantly enriched. Second, the standard environmental drivers used to control SE, i.e. light, growth regulators and cell density, were clearly perceived at the molecular level at different developmental stages. Third, expression profiles of auxin-related genes, transcription factor-related genes and secondary metabolism-related genes were analyzed during SE. Gene co-expression networks were also inferred. Auxin-related genes were upregulated during dedifferentiation and redifferentiation while transcription factor-related genes were switched on from the embryogenic callus and onward. Secondary metabolism-related genes were switched off during dedifferentiation and switched back on at the onset of redifferentiation. Secondary metabolites and endogenous IAA content were tightly linked with their respective gene expression. Lastly, comparing Arabica embryogenic and non-embryogenic cell transcriptomes enabled the identification of biological processes involved in the acquisition of embryogenic capacity. CONCLUSIONS: The present analysis showed that transcript fingerprints are discriminating signatures of cell fate and are under the direct influence of environmental drivers. A total of 23 molecular candidates were successfully identified overall the 12 developmental stages and can be tested in many plant species to optimize SE protocols in a rational way.

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.

The absence of the caffeine synthase gene is involved in the naturally decaffeinated status of Coffea humblotiana, a wild species from Comoro archipelago.

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.

Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee.

Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth statistical analysis performed on 104 metabolites revealed that massive re-configuration of metabolic pathways induced SE. During initial dedifferentiation, a sharp decrease in phenolic compounds and caffeine levels was also observed while auxins, cytokinins and ethylene levels were at their highest. Totipotency reached its highest expression during the callus stages when a shut-off in hormonal and metabolic pathways related to sugar and energetic substance hydrolysis was evidenced. Abscisic acid, leucine, maltotriose, myo-inositol, proline, tricarboxylic acid cycle metabolites and zeatin appeared as key metabolic markers of the embryogenic capacity. Combining metabolomics with multiphoton microscopy led to the identification of chlorogenic acids as markers of embryo redifferentiation. The present analysis shows that metabolite fingerprints are signatures of cell fate and represent a starting point for optimizing SE protocols in a rational way.

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.

Differential regulation of caffeine metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta).

Caffeine is a metabolite of great economic importance, especially in coffee, where it influences the sensorial and physiological impacts of the beverage. Caffeine metabolism in the Coffea species begins with the degradation of purine nucleotides through three specific N-methyltransferases: XMT, MXMT and DXMT. A comparative analysis was performed to clarify the molecular reasons behind differences in caffeine accumulation in two Coffea species, namely Coffea arabica and Coffea canephora var. robusta. Three different genes encoding N-methyltransferase were amplified in the doubled haploid Coffea canephora: CcXMT1, CcMXMT1 and CcDXMT. Six genes were amplified in the haploid Coffea arabica: CaXMT1, CaXMT2, CaMXMT1, CaMXMT2, CaDXMT1, and CaDXMT2. A complete phylogenic analysis was performed to identify specific key amino acids defining enzymatic function for each protein identified. Furthermore, a quantitative gene-expression analysis was conducted on leaves and on maturing coffee beans, simultaneously analyzing caffeine content. In the different varieties analyzed, caffeine accumulation is higher in leaves than in the coffee bean maturation period, higher in Robusta than in Arabica. In Robusta, CcXMT1 and CcDXMT gene expressions are predominant and transcriptional activity is higher in leaves than in maturing beans, and is highly correlated to caffeine accumulation. In Arabica, the CaXMT1 expression level is high in leaves and CaDXMT2 as well to a lesser extent, while global transcriptional activity is weak during bean maturation, suggesting that the transcriptional control of caffeine-related genes differs within different organs and between Arabica and Robusta. These findings indicate that caffeine accumulation in Coffea species has been modulated by a combination of differential transcriptional regulation and genome evolution.

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

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.

Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination.

BACKGROUND: Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. RESULTS: Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. CONCLUSIONS: Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death associated with post-germination of the coffee grain. Expression analysis of the cysteine proteinase inhibitor genes suggests that CcCPI-1 could primarily be involved in modulating the activity of grain CP activity; while CcCPI-4 may play roles modulating grain CP activity and in the protection of the young coffee seedlings from insects and pathogens. CcCPI-2 and CcCPI-3, having lower and more widespread expression, could be more general "house-keeping" CPI genes.

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.

Assessment of DHPLC usefulness in the genotyping of GSTP1 exon 5 SNP: comparison to the PCR-RFLP method.

Single-nucleotide polymorphism (SNP) analysis can be performed by several methods such as PCR-RFLP, real time PCR and mass spectrometry. Denaturating High Pressure Liquid Chromatography (DHPLC) analysis allows the detection of DNA mutations in heteroduplex samples. GSTP1 exon 5 gene presents a single-nucleotide polymorphism (a to g) that results into an amino-acid substitution (Ile to Val). Ile and Val variants are identified respectively by a and b alleles. This polymorphism affects enzyme activity and is highly frequent within Caucasian populations and therefore widely studied in the context of SNP related to cancer susceptibility. Our goal was to evaluate DHPLC usefulness in detecting a well-known SNP in comparison to PCR-RFLP, in the field of molecular epidemiological studies. Fifty Caucasian people were genotyped by both methods. Heterozygous samples were identified easily at two temperatures using the DHPLC method. Discrimination between a/a and b/b homozygous genotypes was done by pooling every homozygous sample with a known a/a sample. Our genotyping using both methods resulted in the characterisation of 32 (64%) a/a homozygous, 18 (36%) a/b heterozygous and 5 (10%) b/b homozygous. All samples were also identically genotyped by the two methods. Our results show that DHPLC is a good alternative to classical PCR-RFLP method in genotyping SNPs. Advantages of this chromatographic method were no restriction site needed and a reduced technical time thanks to an automated injection. Moreover, unlike classical RFLP gel analysis, DHPLC chromatograms provided objective criteria for sample classification.