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Made available in DSpace on 2019-10-06T15:42:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-15 Agropolis Fondation Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Information about population structure and genetic relationships within and among wild and brazilian Coffea arabica L. genotypes is highly relevant to optimize the use of genetic resources for breeding purposes. In this study, we evaluated genetic diversity, clustering analysis based on Jaccard’s coefficient and population structure in 33 genotypes of C. arabica and of three diploid Coffea species (C. canephora, C. eugenioides and C. racemosa) using 30 SSR markers. A total of 206 alleles were identified, with a mean of 6.9 over all loci. The set of SSR markers was able to discriminate all genotypes and revealed that Ethiopian accessions presented higher genetic diversity than commercial varieties. Population structure analysis indicated two genetic groups, one corresponding to Ethiopian accessions and another corresponding predominantly to commercial cultivars. Thirty-four private alleles were detected in the group of accessions collected from West side of Great Rift Valley. We observed a lower average genetic distance of the C. arabica genotypes in relation to C. eugenioides than C. canephora. Interestingly, commercial cultivars were genetically closer to C. eugenioides than C. canephora and C. racemosa. The great allelic richness observed in Ethiopian Arabica coffee, especially in Western group showed that these accessions can be potential source of new alleles to be explored by coffee breeding programs. Instituto Agronômico do Paraná (IAPAR), Rodovia Celso Garcia Cid, 375 Km Centro de Ciências Biológicas Área de Genética e Biologia Molecular Universidade Estadual de Londrina (UEL), CP 10.011 CIRAD UMR AGAP AGAP Univ. Montpellier CIRAD INRA INRIA Montpellier SupAgro Departamento de Agronomia Universidade Estadual de Londrina (UEL), CP 6001 Instituto Agronômico do Paraná (IAPAR) Mestrado em Agricultura Conservacionista, CP 481 Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP) Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA Café) Laboratório de Biotecnologia de Plantas – IAPAR Embrapa Café, Rodovia Celso Garcia Cid, 375 km Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP)

Made available in DSpace on 2018-12-11T17:00:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-02-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Studies in diploid parental species of polyploid plants are important to understand their contributions to the formation of plant and species evolution. Coffeaeugenioides is a diploid species that is considered to be an ancestor of allopolyploid Coffeaarabica together with Coffeacanephora. Despite its importance in the evolutionary history of the main economic species of coffee, no study has focused on C. eugenioides molecular genetics. RNA-seq creates the possibility to generate reference transcriptomes and identify coding genes and potential candidates related to important agronomic traits. Therefore, the main objectives were to obtain a global overview of transcriptionally active genes in this species using next-generation sequencing and to analyze specific genes that were highly expressed in leaves and fruits with potential exploratory characteristics for breeding and understanding the evolutionary biology of coffee. A de novo assembly generated 36,935 contigs that were annotated using eight databases. We observed a total of ~5000 differentially expressed genes between leaves and fruits. Several genes exclusively expressed in fruits did not exhibit similarities with sequences in any database. We selected ten differentially expressed unigenes in leaves and fruits to evaluate transcriptional profiles using qPCR. Our study provides the first gene catalog for C. eugenioides and enhances the knowledge concerning the mechanisms involved in the C. arabica homeologous. Furthermore, this work will open new avenues for studies into specific genes and pathways in this species, especially related to fruit, and our data have potential value in assisted breeding applications. Programa de Pós-Graduação em Genética e Biologia Molecular Universidade Estadual de Londrina (UEL) Laboratório de Biotecnologia Vegetal Instituto Agronômico do Paraná (IAPAR) Laboratório de Genômica e Expressão Departamento de Genética Evolução e Bioagentes Instituto de Biologia Universidade Estadual de Campinas (UNICAMP), Campinas Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) UMR AGAP, 34398 Embrapa Café Department of Botany Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP) Department of Botany Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP) CNPq: 1002-02 PHEGECO

The management of diversity for conservation and breeding is of great importance for all plant species and is particularly true in perennial species, such as the coffee Coffea canephora. This species exhibits a large genetic and phenotypic diversity with six different diversity groups. Large field collections are available in the Ivory Coast, Uganda and other Asian, American and African countries but are very expensive and time consuming to establish and maintain in large areas. We propose to improve coffee germplasm management through the construction of genetic core collections derived from a set of 565 accessions that are characterized with 13 microsatellite markers. Core collections of 12, 24 and 48 accessions were defined using two methods aimed to maximize the allelic diversity (Maximization strategy) or genetic distance (Maximum- Length Sub-Tree method). A composite core collection of 77 accessions is proposed for both objectives of an optimal management of diversity and breeding. This core collection presents a gene diversity value of 0.8 and exhibits the totality of the major alleles (i.e., 184) that are present in the initial set. The seven proposed core collections constitute a valuable tool for diversity management and a foundation for breeding programs. The use of these collections for collection management in research centers and breeding perspectives for coffee improvement are discussed.

Base composition is highly variable among and within plant genomes, especially at third codon positions, ranging from GC-poor and homogeneous species to GC-rich and highly heterogeneous ones (particularly Monocots). Consequently, synonymous codon usage is biased in most species, even when base composition is relatively homogeneous. The causes of these variations are still under debate, with three main forces being possibly involved: mutational bias, selection and GC-biased gene conversion (gBGC). So far, both selection and gBGC have been detected in some species but how their relative strength varies among and within species remains unclear. Population genetics approaches allow to jointly estimating the intensity of selection, gBGC and mutational bias. We extended a recently developed method and applied it to a large population genomic dataset based on transcriptome sequencing of 11 angiosperm species spread across the phylogeny. We found that at synonymous positions, base composition is far from mutation-drift equilibrium in most genomes and that gBGC is a widespread and stronger process than selection. gBGC could strongly contribute to base composition variation among plant species, implying that it should be taken into account in plant genome analyses, especially for GC-rich ones., Author summary In protein coding genes, base composition strongly varies within and among plant genomes, especially at positions where changes do not alter the coded protein (synonymous variations). Some species, such as the model plant Arabidopsis thaliana, are relatively GC-poor and homogeneous while others, such as grasses, are highly heterogeneous and GC-rich. The causes of these variations are still debated: are they mainly due to selective or neutral processes? Answering to this question is important to correctly infer whether variations in base composition may have functional roles or not. We extended a population genetics method to jointly estimate the different forces that may affect synonymous variations and applied it to genomic datasets in 11 flowering plant species. We found that GC-biased gene conversion, a neutral process associated with recombination that mimics selection by favouring G and C bases, is a widespread and stronger process than selection and that it could explain the large variation in base composition observed in plant genomes. Our results bear implications for analysing plant genomes and for correctly interpreting what could be functional or not.

Made available in DSpace on 2018-12-11T17:08:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ∼24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages. Programa de Pós-GraduacEao em Genética e Biologia Molecular Centro de Ciencias Biológicas Universidade Estadual de Londrina (UEL) Laboratório de Biotecnologia Vegetal Instituto Agronómico do Paraná (IAPAR) Laboratório de Genômica e Expressao Departamento de Genética Evolução e Bioagentes Instituto de Biologia Universidade Estadual de Campinas (UNICAMP) Departamento de Botânica Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP) Centre de Coopération Internationale en Recherche Agronomique Pour le Développement UMR AGAP Programa de Pós Graduação em Agronomia Universidade do Oeste Paulista (UNOESTE) Empresa Brasileira de Pesquisa Agropecuária (Embrapa Café) Departamento de Botânica Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP)

SUMMARYControlled irrigation during the dry period associated with adequate nitrogen (N), phosphorous (P) and potassium (K) fertilization led to the accumulation of biochemical compounds in coffee beans considered as positive precursors of beverage quality. Adult plants of coffee (Coffea arabica ‘Rubi’) were cultivated using different water regimes (WR) and fertilization conditions under the dry climate of the Brazilian Cerrado. Coffee-bean physical characteristics were evaluated as well as biochemical composition by near-infrared analysis. The K treatment mostly affected bean biochemistry, lipid and chlorogenic acid (CGA) contents, which increased with increasing amounts of fertilizer. Caffeine contents increased with higher amounts of N, but no significant effects of P treatment on bean biochemical composition were observed. Sucrose and total lipid contents always appeared higher in beans of non-irrigated plants than those beans from plants grown with continuous irrigation. In contrast, caffeine and CGA contents were higher in beans of irrigated as compared with non-irrigated plants. For the first time, the current results showed that controlled management of irrigation during the dry period associated with reasonable NPK fertilization led to the accumulation of biochemical compounds in coffee beans considered as positive precursors of beverage quality.

Made available in DSpace on 2018-12-11T16:45:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Lipids are among the major chemical compounds present in coffee beans, and they affect the flavor and aroma of the coffee beverage. Coffee oil is rich in kaurene diterpene compounds, mainly cafestol (CAF) and kahweol (KAH), which are related to plant defense mechanisms and to nutraceutical and sensorial beverage characteristics. Despite their importance, the final steps of coffee diterpenes biosynthesis remain unknown. To understand the molecular basis of coffee diterpenes biosynthesis, we report the content dynamics of CAF and KAH in several Coffea arabica tissues and the transcriptional analysis of cytochrome P450 genes (P450). We measured CAF and KAH concentrations in leaves, roots, flower buds, flowers and fruit tissues at seven developmental stages (30–240 days after flowering - DAF) using HPLC. Higher CAF levels were detected in flower buds and flowers when compared to fruits. In contrast, KAH concentration increased along fruit development, peaking at 120 DAF. We did not detect CAF or KAH in leaves, and higher amounts of KAH than CAF were detected in roots. Using P450 candidate genes from a coffee EST database, we performed RT-qPCR transcriptional analysis of leaves, flowers and fruits at three developmental stages (90, 120 and 150 DAF). Three P450 genes (CaCYP76C4, CaCYP82C2 and CaCYP74A1) had transcriptional patterns similar to CAF concentration and two P450 genes (CaCYP71A25 and CaCYP701A3) have transcript accumulation similar to KAH concentration. These data warrant further investigation of these P450s as potential candidate genes involved in the final stages of the CAF and KAH biosynthetic pathways. Instituto Agronômico do Paraná (IAPAR) Laboratório de Biotecnologia Vegetal, CP 481 Universidade Estadual de Londrina (UEL), CP 6001 Instituto Agronômico do Paraná (IAPAR) Laboratório de Ecofisiologia Vegetal, CP 481 Centre de Coopération Internationale en Recherche Agronomique Pour le Développement (CIRAD) UMR AGAP Universidade do Oeste Paulista (UNOESTE), Rodovia Raposo Tavares, Km 572 Universidade Estadual Paulista (UNESP) Instituto de Biociências de Rio Claro, Avenida 24-A, 1515 Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA Café) Universidade Estadual Paulista (UNESP) Instituto de Biociências de Rio Claro, Avenida 24-A, 1515

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

Coffea canephora Pierre ex Frohener is a perennial plant originated from Africa. Two main groups, Guinean and Congolese, have already been identified within this species. They correspond to main refugia in western and central Africa. In this paper we present the analysis of a region that has not yet been studied, Uganda. Two wild, one feral (once cultivated but abandoned for many years), and two cultivated populations of C. canephora from Uganda were evaluated using 24 microsatellite markers. Basic diversity, dissimilarity and genetic distances between individuals, genetic differentiation between populations, and structure within populations were analysed. Expected heterozygosity was high for wild compartments (0.48 to 0.54) and for cultivated and feral ones (0.57 to 0.59), with the number of private alleles ranging from 12 for cultivated genotypes to 37 for a wild compartment. The Ugandan samples show significant population structuring. We compared the Ugandan populations with a representative sample of known genetic diversity groups within the species using 18 markers. Coffea canephora of Ugandan origin was found to be genetically different from previously identified diversity groups, implying that it forms another diversity group within the species. Given its large distribution and extremely recent domestication, C. canephora can be used to understand the effect of refugia colonization on genetic diversity.

The exploration of accessions originating from Ethiopia is one option available to increase the genetic basis of new coffee cultivars. The aim of this study was to describe the chemical composition variability of Ethiopian coffee accessions maintained in Londrina and to evaluate the year effect on that. We observed no significant interaction between the years of cultivation and the amounts of caffeine, protein, lipids, chlorogenic acids, sucrose, cafestol and kahweol, thereby suggesting that evaluations of one year were reliable to describe this collection. All compounds showed variability, but the highest variability was found in cafestol and kahweol (32.81 and 34.45 %, respectively). Significant correlations among caffeine, chlorogenic acids and protein as well as between sucrose and lipids were observed. A significant negative correlation was found between kahweol and cafestol, and kahweol had positive and negative correlation with caffeine and chlorogenic acids, respectively. Hierarchical clustering analyses identified groups in the population, and the diversity found in one crop year was similar to that for 3 years. Greater diversity of composition was observed among the Western accessions compared to the ones from the East, but no clear separation was found between the accessions of these regions. These accessions are potential sources to increase the genetic diversity to obtain new coffee cultivars.

Eleven mating designs, 6 top-crosses and 5 North Carolina II, were used to estimate narrow sense, broad sense and within-cross heritability of Coffea canephora yield in Cote d'Ivoire. The objective was to predict achievable genetic gains according to different breeding strategies. Narrow sense, broad sense, family and within-cross heritabilities were 0.32, 0.43, 0.83 and 0.22, on average respectively. The percentage of additivity was high (81% on average). A degree of variation was nevertheless observed for estimates over trials. This was found to be mainly due to parent sampling in each trial. By theoretical computation, the minimum number of parents required to obtain optimum precision was found to be around 30. Predicted genetic gains for yield through clonal selection at a selection level of 1% ranged from 7 to 28% over the best hybrid in each trial, suggesting thus clones still interesting for breeders. The consequences for coffee breeding are discussed.

Genetically-modified coffee clones (GMCs) were presented in a previous work. They were created from a single commercial clone of Coffea canephora Pierre (clone 126). Therefore they all have the same genotype, except for the localization of the transgenic insertions. They synthesize the Bacillus thuringiensis endotoxin Cry1Ac against Leucoptera coffeella Gue´- rin-Me´neville, a secondary pest of C. canephora and one of the main pest of C. arabica in South America. The synthetic Cry1Ac gene is regulated by the EF1a- A1 promoter of Arabidopsis thaliana (L.) Heynh. They were tested in an experimental field for their resistance against the pest insect in a previous work. In the present work, levels of Cry1Ac were measured in the leaves. The insecticidal protein was evenly distributed in all the leaves. Cry1Ac levels were measured once in the coffee plants of a sensitive GMC and of 14 resistant ones grown in the experimental field and in plants grown in a greenhouse. Some resistant GMCs contained higher levels but it is not possible to confirm that it would be enough for a sustainable resistance to the pest. Growth speed was variable in the plot. The correlation with plant height and other indicators of plant growth was examined. Cry1Ac levels in the GMCs were negatively correlated with growth speed. The latter was not statistically influenced neither by Cry1Ac synthesis nor by the genetic modification in itself as seen by comparing the GMCs and the unmodified control clone 126. Hence the conclusion is that the growth conferred to field-grown plants by environmental factors and especially the soil was probably the underlying cause of the negative correlation. Other field experiments would be necessary in order to confirm this result. It would be important that the genetic construct inserted in these GMCs and mainly the EF1a-A1 promoter of the Cry1Ac gene be reconsidered since Cry1Ac levels might be too low to provide efficient and sustainable protection against L. coffeella in a highly favourable environment for coffee plants. Alternatives are discussed.

Coffee, tea, and chocolate converge Caffeine has evolved multiple times among plant species, but no one knows whether these events involved similar genes. Denoeud et al. sequenced the Coffea canephora (coffee) genome and identified a conserved gene order (see the Perspective by Zamir). Although this species underwent fewer genome duplications than related species, the relevant caffeine genes experienced tandem duplications that expanded their numbers within this species. Scientists have seen similar but independent expansions in distantly related species of tea and cacao, suggesting that caffeine might have played an adaptive role in coffee evolution. Science , this issue p. 1181 ; see also p. 1124

We searched for quantitative trait loci (QTL) associated with the palm oil fatty acid composition of mature fruits of the oil palm E. guineensis Jacq. in comparison with its wild relative E. oleifera (H.B.K) Corte´ s. The oil palm cross LM2T x DA10D between two heterozygous parents was considered in our experiment as an intraspecific representative of E. guineensis. Its QTLs were compared to QTLs published for the same traits in an interspecific Elaeis pseudo-backcross used as an indirect representative of E. oleifera. Few correlations were found in E. guineensis between pulp fatty acid proportions and yield traits, allowing for the rather independent selection of both types of traits. Sixteen QTLs affecting palm oil fatty acid proportions and iodine value were identified in oil palm. The phenotypic variation explained by the detected QTLs was low to medium in E. guineensis, ranging between 10% and 36%. The explained cumulative variation was 29% for palmitic acid C16:0 (one QTL), 68% for stearic acid C18:0 (two QTLs), 50% for oleic acid C18:1 (three QTLs), 25% for linoleic acid C18:2 (one QTL), and 40% (two QTLs) for the iodine value. Good marker co-linearity was observed between the intraspecific and interspecific Simple Sequence Repeat (SSR) linkage maps. Specific QTL regions for several traits were found in each mapping population. Our comparative QTL results in both E. guineensis and interspecific materials strongly suggest that, apart from two common QTL zones, there are two specific QTL regions with major effects, which might be one in E. guineensis, the other in E. oleifera, which are independent of each other and harbor QTLs for several traits, indicating either pleiotropic effects or linkage. Using QTL maps connected by highly transferable SSR markers, our study established a good basis to decipher in the future such hypothesis at the Elaeis genus level.

The recurrent selection schemes recommended by CIRAD for 6 tropical tree crops are presented and compared. Breeding programmes are carried out under cooperation between CIRAD and its partners in regions where the crops are grown. The crops are cacao, coffee, rubber tree, oil palm, coconut and eucalyptus. After a short look at the background, the reasons behind the options chosen are given and the main characteristics of the schemes are described and illustrated. The state of progress and some major results are discussed. Over and above the differences linked to plant biology, the genetic diversity available and the type of varietal output, common characteristics are emphasized. The time taken for each generation means simplifying the intercrossing phases between successive breeding cycles, and thus, the parents tested are heterozygous: whenever possible, a clonal varietal output is an additional source of progress. The use of biotechnologies to study diversity is a valuable asset, and their application for early selection (QTL) is also promising. The preponderance of combining ability coexists with marked heterosis, which poses the problem of the latter's origin. As a result, all of the schemes described, but one, are reciprocal recurrent selection schemes.

Breeding of the allogamous Coffea canephora species in Cote d'Ivoire is based, from 1984 onward, on a reciprocal recurrent selection scheme exploiting the good performance of crosses between two genetically distinct populations (‘Guinean’ and ‘Congolese’). Results are presented here on intergroup cross progenies evaluated on individual plant basis over a six year period in three fully randomized trials planted in 1985 and 1986. Yields of the bi-parental crosses varied from 16 to 1407% of the mean of the two commercial clonal cultivars and the most productive crosses were also the most vigourous. Selection of individual trees and cross progenies, based on a combined individual-family index, was carried out for high yield, good vigour of young plants and moderate canopy diameter of adult trees. Expected genetic gains, as estimated in relation to the mean of all crosses, for selection of 5% of the best trees, were high for yield (over 60%), moderate for young plant vigour (14 to 18%), and, as intended, low for canopy diameter. In addition, selection by independent culling for leaf rust resistance and bean weight was carried out. As a result, 6 promising crosses with yield similar or higher than those of the control cultivars, were selected as candidates for commercial use, as well as 65 individual trees, within 19 different crosses. Expected genetic gain for yield for these selected trees is about 60% compared to the average yield of the most productive control clone. Continuation of the programme is discussed, with specific attention to further progress expected for quality traits.

Background A reciprocal recurrent selection program has been under way for the Coffea canephora coffee tree for approximately thirty years in the Ivory Coast. Association genetics would help to speed up this program by more rapidly selecting zones of interest in the genome. However, prior to any such studies, the linkage disequilibrium (LD) needs to be assessed between the markers on the genome. These data are essential for guiding association studies. Results This article describes the first results of an LD assessment in a coffee tree species. Guinean and Congolese breeding populations of C. canephora have been used for this work, with the goal of identifying ways of using these populations in association genetics. We identified changes in the LD along the genome within the different C. canephora diversity groups. In the different diversity groups studied, the LD was variable. Some diversity groups displayed disequilibria over long distances (up to 25 cM), whereas others had disequilibria not exceeding 1 cM. We also discovered a fine structure within the Guinean group. Conclusions Given these results, association studies can be used within the species C. canephora. The coffee recurrent selection scheme being implemented in the Ivory Coast can thus be optimized. Lastly, our results could be used to improve C. arabica because one of its parents is closely related to C. canephora.

As the second species used for commercial coffee, evaluation of Coffea canephora Pierre ex Froehner population genetics is a challenging task for coffee breeding. This study examined the pattern of genetic variability and genetic relationships of cultivated and wild populations of C. canephora sampled across the Guineo-Congolese region of Africa and some improved populations maintained in field genebanks. A total of 293 individuals, sampled from 17 populations, were genotyped with 39 nuclear microsatellite markers. Genetic diversity and structure were investigated with both a model-based and a graphical approach; isolation by distance was also tested. Relationships between the diversity clusters are discussed with regard to differentiation due to several glacial refuges during the Last Glacial Maximum (LGM). High genetic diversity within C. canephora is confirmed with a mean number of alleles of 11.85 per marker, a mean gene diversity of 0.72 and a mean observed heterozygosity of 0.36. An overall structure of two main groups (Guinean and Congolese) subdivided in six subgroups (2 for the Guinean and 4 for the Congolese) was found, including one described for the first time in the Guinean group. A fine structure within the Guinean group was also newly detected. Genetic structure of C. canephora appears to be consistent with its geographic repartition at the continent scale. Structure of diversity was found in accordance with localizations of refuge zones during LGM and migration from this period. Results from this genetic structure study raise our capabilities to better manage and use the collections of genetics resources for breeding purposes. Those results will be used in future association studies to optimize the number of genotypes to be phenotyped.

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.

IATE Axe 4 : Biotechnologie microbienne et enzymatique des lipides et des agropolymères; We chose an Elaeis interspecific pseudo-backcross of first generation (E. oleifera x E. guineensis) x E. guineensis to identify quantitative trait loci (QTLs) for fatty acid composition of palm oil. A dense microsatellite linkage map of 362 loci spanned 1.485 cM, representing the 16 pairs of homologous chromosomes in the Elaeis genus from which we traced segregating alleles from both E. oleifera and E. guineensis grandparents. The relative linear orders of mapped loci suggested the probable absence of chromosome rearrangements between the E. oleifera and E. guineensis genomes. A total of 19 QTL associated to the palm oil fatty acid composition were evidenced. The QTL positions and the species origin as well as the estimated effects of the QTL marker alleles were in coherence with the knowledge of the oil biosynthesis pathway in plants and with the individual phenotypic correlations between the traits. The mapping of chosen Elaeis key genes related to oleic acid C18:1, using intra-gene SNPs, supported several QTLs underlying notably FATA and SAD enzymes. The high number of hyper-variable SSR loci of known relative linear orders and the QTL information make these resources valuable for such mapping study in other Elaeis breeding materials.

Knowledge on heritability is essential for selecting varieties resistant against coffee wilt disease caused by Fusarium xylarioides, which is currently devastating coffee in East and Central Africa. Variability of the resistance against coffee wilt disease in Coffea canephora and its inheritance were investigated in three experiments corresponding to a clonal trial involving 20 clones, a 10- parent partial diallel progeny, and a half-sib progeny test. There were significant quantitative genetic variations among the clones and progenies, suggesting polygenic control of the resistance. Around 50-65 % tree mortality was the optimal disease level for calculating heritability and genetic gains. General and specific combining abilities calculated within the optimal disease range for partial diallel analysis were significant. Broad-sense heritability for the same analysis and same disease range was moderate (0.329), and corresponding narrow-sense heritability was low (0.112). Broad-sense heritability from clones in the field at the same disease range was also moderate (0.333). Narrow-sense heritability from regression of half-sib progeny means onto parent means in the field and screen house was 0.183 and 0.369, respectively. Selecting tolerant clones for improvement against the disease is possible, and genetic gains are possible by using tolerant parents in breeding programs.

UMR 1334 AGAP : Equipe AFEF 'Architecture et Fonctionnement des Espèces fruitières' ; Team AFFS 'Architecture and Functioning of Fruit Species' Contact: valerie.pujade-renaud@cirad.fr; Corynespora Leaf Fall (CLF) is a major disease of rubber tree (Hevea brasiliensis) caused by the Ascomycota Corynespora cassiicola. Here we describe the cloning and characterization of a gene encoding cassiicolin (Cas), a glycosylated cystein-rich small secreted protein (SSP) identified as a potential CLF disease effector in rubber tree. Three isolates with contrasted levels of aggressiveness were analyzed comparatively. The cassiicolin gene was detected - and the toxin successfully purified - from the isolates with high and medium aggressiveness (CCP and CCAM3 respectively) but not from the isolate with the lowest aggressiveness (CCAM1), suggesting the existence of a different disease effector in the later. CCP and CCAM3 carried strictly identical cassiicolin genes and produced toxins of identical mass, as evidence by mass spectrometry analysis, thus suggesting conserved post-translational modifications in addition to sequence identity. The differences in aggressiveness between CCP and CCAM3 may be attributed to differences in cassiicolin transcript levels rather than qualitative variations in cassiicolin structure. Cassiicolin may play an important role in the early phase of infection since a peak of cassiicolin transcripts occurred in 1 or 2 days after inoculation (before the occurrence of the first symptoms), in both the tolerant and the susceptible cultivars.

Background Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. Results The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. Conclusion We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.

Coffea canephora breeding requires combining sustainable productivity with improved technological and cup quality characteristics. Beverage quality is a complex and subjective trait, and breeding for this trait is time consuming and depends on knowledge of the genetics of its components. A highly variable C. canephora progeny resulting from an intraspecific cross was assessed for 63 traits over 5 years. To identify quantitative trait loci (QTLs) controlling agronomic, technological, and quality-related traits, a genetic map comprising 236 molecular markers was constructed, and composite interval mapping was performed. Beverage quality was evaluated in relation to biochemical and cup tasting traits. QTLs were identified for almost half of the traits evaluated, with effects ranging from 6% to 80% of phenotypic variation. Most of them present a consistent detection over years. The strongest QTLs explained a high percentage of the variation for yield in 2006 (34% to 57%), bean size (25% to 35%), content of chlorogenic acids (22% to 35%), sucrose and trigonelline content (29% to 81%), and acidity and bitterness of coffee beverages (30% to 55%). Regions of the C. canephora genome influencing beverage quality were identified. Five QTL zones were co-localized with candidate genes related to the biosynthesis of the analyzed traits: two genes coding for caffeine biosynthesis, one gene implicated in the biosynthesis of chlorogenic acids, and two genes implicated in sugar metabolism. This is one of the first studies on the identification of QTLs combining agronomic and quality traits in coffee. The high variability of quality traits within C. canephora and the presence of consistent QTLs offer breeders a promising tool to improve coffee cup quality.

Genetic parameters were estimated in trials planted in connection with a reciprocal recurrent selection programme applied to Coffea canephora in Cote d'Ivoire. Narrow sense heritability values, estimated from a breakdown of the analysis of variance, were high for architectural characters (0.22 to 0.78) and medium for tree vigour (0.13 to 0.40) and for bean weight (0.15 to 0.28). Calculated heritability values for the first two harvests were high (h2>0.7), but much lower for the following harvests (less than 0.2). Heritability estimates by parent-offspring regressions gave variable results, though they were often similar to those obtained by variance analysis, especially for bean weight. Phenotypic and genetic correlations between tree vigour traits and productivity were high. Bean weight was not correlated with vigour or production. Canopy diameter of 4 year-old trees was closely correlated with cumulated productivity from 2 to 5 years in the absence of development competition between trees, but the correlation was low if trees were in competition with each other. The consequences of the results for the choice of characters to be selected among intergroup hybrids or the parents of both populations are discussed.

Two breeding populations have been identified with Coffea canephora, based on geographical and genetic differences: the Guinean group from West Africa and the Congolese group from central Africa. A reciprocal recurrent selection programme based on these groups was initiated in Cote d'Ivoire in 1984. Genotypes of both groups available in field collections are either of cultivated or wild origin. Genotypic and phenotypic variability was evaluated within and between the groups for the following characteristics: isozyme patterns, architecture, drought tolerance and vigour, technological and organoleptic traits, pest and disease resistance. Variation between and within groups was found to be large. Based on isozyme analysis and phenotypic observations, two subgroups were identified within the Congolese group. Phenotypic values of parents and testers used in the first cycle of reciprocal recurrent selection are presented. Results of two intergroup hybrid trials indicate high yield and vigour for these hybrids compared to intragroup hybrids, justifying the reciprocal selection approach. Furthermore, the variation observed indicates that recurrent selection would allow progress for selection traits for both populations and intergroup hybrids.

La présente étude montre la transférabilité de marqueurs microsatellites développés sur les deux espèces de caféiers cultivées (Coffea arabica L. et C. canephora Pierre ex Froehn.) à 15 espèces représentant les principaux groupes précédemment identifiés du genre Coffea. L'évaluation de la diversité et des ressources génétiques disponibles au sein du genre Coffea et le développement de marqueurs moléculaires transférables d'une espèce à l'autre sont des étapes importantes pour l'amélioration de ces deux espèces. Nous avons travaillé sur 15 espèces avec 60 marqueurs microsatellites développés suivant différentes méthodologies (banques enrichies en microsatellites, banques BAC, séquences de gènes). Nous avons plus particulièrement analysé quatre espèces d'intérêt en commerce ou en amélioration. Nos résultats établissent que les microsatellites sont hautement transférables dans le genre Coffea. Nous mettons en évidence l'important réservoir de diversité pour l'amélioration que constituent les espèces sauvages de ce genre. Enfin nous discutons des implications pour de futures études de cartographie comparée et l'amélioration des deux espèces cultivées.

International audience; Coffea canephora Pierre breeding in Cote d'Ivoire is based on indirect reciprocal recurrent selection (RRSi) using the Congolese and Guinean populations. In practice, the genotypes of each population (78 Congolese genotypes and 100 Guinean genotypes) are tested with several testers from the reciprocal population: two Guinean and three Congolese testers. These testers are heterozygous as C. canephora is a strictly outcrossing species. After a first cycle of RRSi, we judged the efficiency of these testers for yield, susceptibility to coffee leaf rust (SCLR) and hundred bean weight (HBW). All the testers discriminated the tested genotypes highly significantly (P < 0.01), even though differences were found in the degrees of discrimination. For yield and SCLR, no interaction between tested genotypes and testers was observed and correlations between test values obtained from different testers were significant or highly significant. Furthermore, test values of tested genotypes could be used to predict the yield and SCLR of between-population (BP) hybrids. Prediction was always more accurate with the best tester than with the mean of several testers. As opposed to yield and SCLR, HBW showed significant interactions between tested genotypes and testers and HBW of hybrids could not be predicted from the test value of the genotypes. We conclude that only one reciprocal tester can be used in the next cycles of RRSi applied to C. canephora. This will allow either a reduced cost of selection or increased selection intensity.

Soixante-dix pour cent des 103 espèces de Coffea actuellement identifiées sont menacées d'extinction, mais peu d'entre elles sont étudiées. Un jeu de 40 marqueurs microsatellites polymorphes a été développé en utilisant une banque génomique de Coffea canephora enrichie en GA/GT. L'amplification de ces marqueurs a été testée sur des accessions de C. heterocalyx (une espèce déclarée " Gravement Menacée ") et C. pseudozanguebariae (une espèce déclarée " Vulnérable "), appartenant à différents clades géographiques Africains. Tous les microsatellites sont polymorphes chez C. canephora, avec un nombre moyen d'allèles par locus de plus de 3 (sur un minimum de 9 génotypes testés). Les hétérozygoties observées et attendues, calculées pour les espèces C. canephora et C. pseudozanguebariae varient respectivement entre 0,10 et 0,91; et entre 0,20 et 0,77. Au total, 95 % des paires d'amorces ont permis une amplification chez C. heterocalyx et C. pseudozanguebariae, indiquant un fort taux de transfert au sein du genre Coffea. Ce large jeu de marqueurs sera utile pour des études génétiques approfondies des espèces menacées de Coffea.

To understand the importance of ethylene receptor genes in the quality of coVee berries three full-length cDNAs corresponding to a putative ethyl- ene receptor gene (ETR1) were isolated from CoVea canephora cDNA libraries. They diVered by their 3UTR and contained a main ORF and a 5UTR short ORF putatively encoding a small polypeptide. The CcETR1 gene, present as a single copy in the C. cane- phora genome, contained Wve introns in the coding region and one in its 5UTR. Alternative splicing can occur in C. canephora and C. pseudozanguebariae, lead- ing to a truncated polypeptide. C. pseudozanguebariae ETR1 transcripts showed various forms of splicing alterations. This gene was equally expressed at all stages of fruit development. A segregation study on an inter-speciWc progeny showed that ETR1 is related to the fructiWcation time, the caVeine content of the green beans, and seed weight. Arabidopsis transformed etio- lated seedlings, which over-expressed CcETR1, dis- played highly reduced gravitropism, but the triple response was observed in an ethylene enriched envi- ronment. These plants behaved like a low-concentra- tion ethylene-insensitive mutant thus conWrming the receptor function of the encoded protein. This gene showed no induction during the climacteric crisis but some linkage with traits related to quality.

Corresponding author: E-mail: thierry.leroy@cirad.fr; International audience; The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productiveand confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.

International audience; A pluriannual field trial of transgenic clones of Coffea canephora (the Robusta coffee tree) transformed for resistance to the lepidopteran coffee leaf miner Leucoptera coffeella was installed in French Guiana. Fifty-eight transformed clones produced by transformation of the C. canephora clone 126 were planted. They were harbouring the pEF1alpha constitutive promoter of Arabidopsis thaliana controlling either the Bacillus thuringiensis native gene for the cry1Ac insecticidal protein (eight clones) or a synthetic cry1Ac gene (53 clones). The vectors for the transformation were a strain of the bacterium Agrobacterium tumefaciens and one of Agrobacterium rhizogenes. The transformed clones were generally independent, presenting different integration patterns of the genetic construct. Four randomly distributed groups of five plants per transformed clone were planted along with 60 untransformed control trees. Over a 4-year period after plantation six releases of L. coffeella were performed. Mines on the leaves are the marks of larvae development and were counted on plants. A majority of the independent transformed clones harbouring the synthetic gene and transformed by the strain of A. tumefaciens displayed constantly much less mines than the control, therefore expressing a stable resistance. The need for complementary research is presented

A factorial crossing scheme of #Coffea canephora# (two parents from the Congolese group crossed to 14 parents of the Guinean group) was used to evaluate genetic parameters of several biochemical compounds, bean weight and crop outturn (ration of dry bean weight to fresh berry weight). For most characters studied, additive genetic effects were preponderant. Narrow-sense heritability was high for caffeine content (h2(ns): 0.80), fat matter content (h2(ns): 0.74), bean weight (h2(ns): 0.73) and crop outturn (h2(ns): 1). It was intermediate for trigonelline (h2(ns): 0.38) and chlorogenic acid (h2(ns): 0.36) content. Only sucrose content had a low narrow-sense heritability (h2(ns): 0.11 ). There were few genetic and environmental correlations. Consequences for breeding, in relation to coffee drinking quality, are discussed. (Resume d'auteur)

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.