A single polyploidization event at the origin of the tetraploid genome of Coffea arabica is responsible for the extremely low genetic variation in wild and cultivated germplasm.

The genome of the allotetraploid species Coffea arabica L. was sequenced to assemble independently the two component subgenomes (putatively deriving from C. canephora and C. eugenioides) and to perform a genome-wide analysis of the genetic diversity in cultivated coffee germplasm and in wild populations growing in the center of origin of the species. We assembled a total length of 1.536 Gbp, 444 Mb and 527 Mb of which were assigned to the canephora and eugenioides subgenomes, respectively, and predicted 46,562 gene models, 21,254 and 22,888 of which were assigned to the canephora and to the eugeniodes subgenome, respectively. Through a genome-wide SNP genotyping of 736 C. arabica accessions, we analyzed the genetic diversity in the species and its relationship with geographic distribution and historical records. We observed a weak population structure due to low-frequency derived alleles and highly negative values of Taijma's D, suggesting a recent and severe bottleneck, most likely resulting from a single event of polyploidization, not only for the cultivated germplasm but also for the entire species. This conclusion is strongly supported by forward simulations of mutation accumulation. However, PCA revealed a cline of genetic diversity reflecting a west-to-east geographical distribution from the center of origin in East Africa to the Arabian Peninsula. The extremely low levels of variation observed in the species, as a consequence of the polyploidization event, make the exploitation of diversity within the species for breeding purposes less interesting than in most crop species and stress the need for introgression of new variability from the diploid progenitors.

DNA extraction and analysis from processed coffee beans.

The authenticity of coffee is an important issue for both producers and consumers. Premium Arabica material is especially prone to being adulterated, and a number of different techniques have been employed to determine the quality of both roasted and instant coffee. Currently, assessment of coffee authenticity relies on chemical methods which can discriminate between coffee species, but not varieties. Several genetic markers are available for assessing coffee origin, but their suitability to testing commercial coffee is limited by the ability to extract DNA from highly processed beans. In this paper, we demonstrate that PCR-grade DNA may be obtained from roasted beans and even instant coffee. This would allow analysis of commercial samples, provided that suitable markers for species/variety identification are found.

Functional characterization of three Coffea arabica L. monoterpene synthases: insights into the enzymatic machinery of coffee aroma.

The chemical composition of the coffee beverage is extremely complex, being made up of hundreds of volatile and non-volatile compounds, many of which are generated in the thermal reactions that occur during the roasting process. However, in the raw coffee bean there are also compounds that survive roasting and are therefore extracted into the beverage. Monoterpenes are an example of this category, as their presence has been reported in the coffee flower, fruit, seed, roasted bean and in the beverage aroma. The present work describes the isolation, heterologous expression and functional characterization of three Coffea arabica cDNAs coding for monoterpene synthases. RNA was purified from C. arabica (cv. Catuai Red) flowers, seeds and fruits at 4 successive ripening stages. Degenerate primers were designed on the most conserved regions of the monoterpene synthase gene family, and then used to isolate monoterpene synthase-like sequences from the cDNA libraries. After 5'- and 3'-RACE, the complete transcripts of 4 putative C. arabica monoterpene synthases (CofarTPS) were obtained. Gene expression in different tissues and developmental stages was analysed. After heterologous expression in Escherichia coli, enzyme activity and substrate specificity were evaluated in vitro by incubation of the recombinant proteins with geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), precursors respectively of mono-, di- and sesquiterpenes. The reaction products were characterized by HS-SPME GC-MS. CofarTPS1 was classified as a limonene synthase gene, while CofarTPS2 and 3 showed lower activity with the production of linalool and ß-myrcene.

Differential responses of Coffea arabica L. leaves and roots to chemically induced systemic acquired resistance.

Coffea arabica is susceptible to several pests and diseases, some of which affect the leaves and roots. Systemic acquired resistance (SAR) is the main defence mechanism activated in plants in response to pathogen attack. Here, we report the effects of benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH), a SAR chemical inducer, on the expression profile of C. arabica. Two cDNA libraries were constructed from the mRNA isolated from leaves and embryonic roots to create 1587 nonredundant expressed sequence tags (ESTs). We developed a cDNA microarray containing 1506 ESTs from the leaves and embryonic roots, and 48 NBS-LRR (nucleotide-binding site leucine-rich repeat) gene fragments derived from 2 specific genomic libraries. Competitive hybridization between untreated and BTH-treated leaves resulted in 55 genes that were significantly overexpressed and 16 genes that were significantly underexpressed. In the roots, 37 and 42 genes were over and underexpressed, respectively. A general shift in metabolism from housekeeping to defence occurred in the leaves and roots after BTH treatment. We observed a systemic increase in pathogenesis-related protein synthesis, in the oxidative burst, and in the cell wall strengthening processes. Moreover, responses in the roots and leaves varied significantly.

Low variability of the protein species synthesized byDrosophila melanogaster embryos.

Stainable proteins as well as newly synthesized polypeptide chains of proteins extracted fromDrosophila melanogaster embryos were analyzed by two-dimensional gel electrophoresis. The following developmental stages were studied: unfertilized eggs, early nuclear multiplication (25 min average age), late nuclear multiplication (105 min), cellular blastoderm (165 min), gastrula (4 h), mesodermal segmentation (6 h) and muscleattachment (8 h). One hundred and fifty stainable spots were present at all developmental stages and were all also synthesized during development, with the exception of 5 unkown proteins and the three yolk proteins. Out of 400 proteins which were labelled by35S-methionine, only 5% showed a reproducible pattern of variable synthesis. Three proteins appeared upon fertilization. The early nuclear multiplication stage showed the largest number of labelled spots while the lowest number of labelled spots was observed at blastoderm formation. The pattern of synthesis of a few specific proteins was also followed. Actin I was synthesized only at 8 h, actin II and actin III were synthesized at all stages. ß-tubulin was synthesized at all stages, while we observed a reduction, if not a cessation, of synthesis of α-tubulin at 105 min, 165 min and 4 h of development. Non heat-shock embryos synthesized a large amount of heat-shock protein (hsp) 84 at 25 min while hsp 70 and 68 were first detected after 4 h of development. Though it is generally accepted that the embryonic genome is activated at blastoderm formation we did not observe a parallel increase in protein species. It is possible that protein synthesis on the new transcripts is below the detection limit of the technique. Alternatively the embryonic messages may gradually substitute preexisting maternal messages or only become available for translation some time after they are transcribed.