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Coffee is one of the world’s largest traded commodities, produced in over 60 countries. Root-knot nematodes (Meloidogyne spp.) have become a major threat in all major coffee-growing areas. So far, more than fifteen species of Meloidogyne have been reported as pathogens of coffee (Coffea arabica L.). Nematocide treatments are only effective in the short term, expensive and environmentally hazardous. Growing nematode-resistant coffee trees constitutes so far the most promising option to control the pest. During the last decade, resistance to root-knot nematode have been identified in spontaneous accessions and relative diploid species. With the aim of improving the resistance to root-knot nematodes of coffee varieties grown in Latin America, a project was initiated in February 2002 with the financial support of the European Community (INCO, Contract ICA4-CT-2001-10070). The selected strategy relies upon the combined development of (i) varieties (either cultivar or rootstock) associating complementary well-characterised resistance genes, and (ii) optimised variety-deployment strategies based on a careful characterisation (geographical distribution, virulence and pathogenicity) of root-knot nematodes populations damaging coffee trees.

Lines of Coffea arabica derived from the Timor Hybrid (hybrid between C. arabica and C. canephora) are resistant to coffee leaf rust (Hemileia vastatrix) and to the nematode Meloidogyne exigua. The introgression of C. canephora resistance genes is suspected of causing a drop in beverage quality. Coffee samples from pure lines, compared in a Trial 1, and from F1 hybrids and parental lines from a half-diallel trial in a Trial 2, were studied for beverage quality, chemical composition and amount of introgressed genetic material. Chemical analyses (caffeine, chlorogenic acids, fat, trigonelline, sucrose) were carried out with near-infrared spectrometry by reflectance of green coffee. The number of amplified fragment length polymorphic (AFLP) markers introgressed from the Timor Hybrid varied from 1 to 37 for the lines studied. There were significant differences between lines for all of the biochemical compounds analysed and for the acidity and the overall standard of the beverage. Two lines (T17927, T17924) were significantly poorer than the controls for sucrose and beverage acidity. T17924 also had more chlorogenic acids and was poorer for the overall standard. However, two highly introgressed lines, T17934 and T17931 (25 and 30 AFLP markers, respectively), did not differ from the non-introgressed controls. There were no correlations between the number of AFLP markers and the chemical contents or beverage attributes. Significant correlations were found between the performance of the parents and their general combining ability for beverage quality. It was concluded that it should be possible to find lines with both the desired resistance genes and good beverage quality. Selection can avoid accompanying the introgression of resistance genes with a drop in beverage quality.

BACKGROUND AND AIMS: The aims of this study were to set up proliferation conditions for hairy roots of Coffea arabica regenerated after transformation by Agrobacterium rhizogenes strain A4-RS, and to carry out the morphological and molecular characterization of hairy root clones maintained over the long term. METHODS: Auxin supply, light conditions and sucrose concentration were modified with the aim of establishing efficient root proliferation conditions. The morphological variability among 62 established hairy root clones was phenotyped by scanning the roots and analysing the images using 'whinRHIZO' software procedures. PCR analysis of integration in transformed root cells of rol and aux oncogenes from the T-DNA of the Ri plasmid was used to study the molecular variability among clones. KEY RESULTS: Auxin supply was necessary to obtain and stimulate growth and branching, and IBA applied at 0·5 μM was the most efficient auxin. Significant differences were shown among the 62 clones for total root length and for the percentage of fine roots. These variables were stable across subcultures and could hence be used for efficient characterization of hairy root clones. The majority of hairy root clones (86 %) exhibited non-significant phenotype differences with non-transformed roots. Eight clones were significantly different from the non-transformed controls in that they possessed a low proportion of fine roots. Two other hairy root clones grew significantly faster than the other clones. The PCR analysis revealed a low variability in the integration of rol and aux oncogenes in transformed root cells. The TR-DNA was never integrated as aux1 and aux2 genes were not found, although rolB and rolC genes from the TL-DNA were always present. CONCLUSIONS: The discovery of low morphological variability among coffee hairy roots together with the identification of morphological variables allowing easy identification of phenotypically altered clones represent two important results. They make hairy roots a possible, and efficient, tool for functional-genomic studies of coffee root genes.

Molecular markers were used to assess polymorphism between and within the genetic bases of coffee (i.e. Typica and Bourbon) spread from Yemen since the early 18th century that have given rise to most arabica cultivars grown world-wide. Eleven Coffea arabica accessions derived from the disseminated bases were evaluated by amplified fragment length polymorphism (AFLP) using 37 primer combinations and simple-sequence repeats (SSRs) produced by six microsatellites. Four cultivars growing in Yemen and 11 subspontaneous accessions collected in the primary centre of diversity of the species were included in the study in order to define their relationship with the accessions derived from the genetic bases of cultivars. One hundred and seven AFLP markers were used to calculate genetic distances and construct a dendrogram. The accessions derived from the disseminated bases were grouped separately, according to their genetic origin, and were distinguished from the subspontaneous accessions. The Yemen cultivars were classified with the Typica-derived accessions. Except for one AFLP marker, all AFLP and SSR markers present in the cultivated accessions were also detected in the subspontaneous accessions. Polymorphism among the subspontaneous accessions was much higher than among the cultivated accessions. It was very low within the genetic bases, confirming the historical documentation on their dissemination. The results enabled a discussion of the genetic diversity reductions that successively occurred during the dissemination of C. arabica from its primary centre of diversity.

The possibility of rapid validation and functional analysis of nematode resistance genes is a common objective for numerous species and particularly for woody species. In this aim, we developed an Agrobacterium rhizogenes-mediated transformation protocol for Coffea arabica enabling efficient and rapid regeneration of transformed roots from the hypocotyls of germinated zygotic embryos, and the subsequent production of composite plants. The A. rhizogenes strain A4RS proved to be the most virulent. High transformation efficiencies (70%) were obtained using a 2-week co-cultivation period at a temperature of 15-18°C. Using a p35S-gusA-int construct inserted in the pBIN19 binary plasmid, we could estimate that 35% of transformed roots were GUS positive (co-transformed). Using the GUS assay as visual marker, 40% composite plants bearing a branched co-transformed rootstock could be obtained after only 12 weeks without selection with herbicides or antibiotics. Transgenic coffee roots obtained with A. rhizogenes did not exhibit the 'hairy' disturbed phenotype and were morphologically similar to normal roots. PCR analyses demonstrated that all co-transformed roots were positive for the expected rolB and gusA genes. Transformed and non-transformed root systems from both susceptible and resistant varieties were inoculated with Meloidogyne exigua nematode individuals. Inoculation of composite plants from the Caturra susceptible variety resulted in the normal development of nematode larvae. Numbers of extracted nematodes demonstrated that transformed roots retain the resistance/sensibility phenotype of varieties from which they are derived. These results suggest that composite plants constitute a powerful tool for studying nematode resistance genes.

Previous study on food plants has shown that near infrared (NIR) spectral methods seem effective for authenticating coffee varieties. We confirm that result, but also show that inter-variety differences are not stable from one harvest to the next. We put forward the hypothesis that the spectral signature is affected by environmental factors. The purpose of this study was to find a way of reducing this environmental variance to increase the method's reliability and to enable practical application in breeding. Spectral collections were obtained from ground green coffee samples from multilocation trials. Two harvests of bean samples from 11 homozygous introgressed lines, and the cv 'Caturra' as the control, supplied from three different sites, were compared. For each site, squared Euclidean distances among the 12 varieties were estimated from the NIR spectra. Matrix correlation coefficients were assessed by the Mantel test. We obtained very good stability (high correlations) for inter-variety differences across the sites when using the two harvests data. If only the most heritable zones of the spectrum were used, there was a marked improvement in the efficiency of the method. This improvement was achieved by treating the spectrum as succession of phenotypic variables, each resulting from an environmental and genetic effect. Heritabilities were calculated with confidence intervals. A near infrared spectroscopy signature, acquired over a set of harvests, can therefore effectively characterize a coffee variety. We indicated how this typical signature can be used in breeding to assist in selection.

Nineteen arabica coffee introgression lines ([BC.sub.1][F.sub.4]) and two accessions derived from a spontaneous interspecific cross (i.e. Timor Hybrid) between Coffea arabica (2n = 4x = 44) and C. canephora (2n = 2x = 22) were analysed for the introgression of C. canephora genetic material. The Timor Hybrid-derived genotypes were evaluated by AFLP, using 42 different primer combinations, and compared to 23 accessions of C. arabica and 8 accessions of C. canephora. A total of 1062 polymorphic fragments were scored among the 52 accessions analysed. One hundred and seventy-eight markers consisting of 109 additional bands (i.e. introgressed markers) and 69 missing bands distinguished the group composed of the Timor Hybrid-derived genotypes from the accessions of C. arabica. AFLP therefore seemed to be an extremely efficient technique for DNA marker generation in coffee as well as for the detection of introgression in C. arabica. The genetic diversity observed in the Timor Hybrid-derived genotypes appeared to be approximately double that in C. arabica. Although representing only a small proportion of the genetic diversity available in C. canephora, the Timor Hybrid obviously constitutes a considerable source of genetic diversity for arabica breeding. Analysis of genetic relationships among the Timor Hybrid-derived genotypes suggested that introgression was not restricted to chromosome substitution but also involved chromosome recombinations. Furthermore, the Timor Hybrid-derived genotypes varied considerably in the number of AFLP markers attributable to introgression. In this way, the introgressed markers identified in the analysed arabica coffee introgressed genotypes were estimated to represent from 9% to 29% of the C. canephora genome. Nevertheless, the amount of alien genetic material in the introgression arabica lines remains substantial and should justify the development of adapted breeding strategies.

Genetic diversity was studied using RAPD markers among119 coffee (Coffea arabica L.) individuals representing 88 accessions derived from spontaneous and subspontaneous trees in Ethiopia, the primary centre of species diversity, six cultivars grown locally in Ethiopia, and two accessions derived from the genetic populations Typica and Bourbon, spread in the 18^sup th^ century, which gave rise to the most currently grown cultivars. Twenty-nine polymorphic fragments were used to calculate a similarity index and construct dendrograms. The Ethiopian material was separated from the Typica- and Bourbon-derived accessions and classified in four groups: one with most of the collected material from southwestern Ethiopia and three from southern and southeastern Ethiopia. Almost all detected diversity was found in the southwestern group while the southern and southeastern groups presented only 59% of identified markers. The genetic distances were low between the southwestern group and the southern and southeastern groups, and between the southwestern group and the Typica- and Bourbon-derived accessions. The cultivated coffee derived from the genetic populations Typica and Bourbon appeared little differentiated from wild coffee growing in the southwest. The results supported the hypothesis that southwestern Ethiopian coffee trees could have been introduced recently in the south and southeast. A separate analysis of the 80accessions classified in the southwestern group allowed identifying particular spontaneous- and subspontaneous-derived accessions and redundancies in the collected material from southwestern Ethiopia. RAPD markers did not detect any within-collection polymorphism except for two trees that were identified as off-types in the CATIE field genebank.[PUBLICATION ABSTRACT]

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.