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

Background: Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, includingCoffea arabica.Results: We identified the conditions required for successful long-term proliferation of embryogenic cultures inC. arabicaand designed a highly efficient and reliableAgrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD₆₀₀= 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.Conclusion: Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results makeAgrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

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

Background: Most disease-resistance (R) genes in plants encode NBS-LRR proteins and belong to one of the largest and most variable gene families among plant genomes. However, the specific evolutionary routes of NBS-LRR encoding genes remain elusive. Recently in coffee tree (Coffea arabica), a region spanning the S _H 3 locus that confers resistance to coffee leaf rust, one of the most serious coffee diseases, was identified and characterized. Using comparative sequence analysis, the purpose of the present study was to gain insight into the genomic organization and evolution of the S _H 3 locus. Results: Sequence analysis of the S _H 3 region in three coffee genomes, E^a and C^a subgenomes from the allotetraploid C. arabica and C^c genome from the diploid C. canephora, revealed the presence of 5, 3 and 4 R genes in E^a, C^a, and C^c genomes, respectively. All these R-gene sequences appeared to be members of a CC-NBS-LRR (CNL) gene family that was only found at the S _H 3 locus in C. arabica. Furthermore, while homologs were found in several dicot species, comparative genomic analysis failed to find any CNL R-gene in the orthologous regions of other eudicot species. The orthology relationship among the S _H 3-CNL copies in the three analyzed genomes was determined and the duplication/deletion events that shaped the S _H 3 locus were traced back. Gene conversion events were detected between paralogs in all three genomes and also between the two sub-genomes of C. arabica. Significant positive selection was detected in the solvent-exposed residues of the S _H 3-CNL copies. Conclusion: The ancestral S _H 3-CNL copy was inserted in the S _H 3 locus after the divergence between Solanales and Rubiales lineages. Moreover, the origin of most of the S _H 3-CNL copies predates the divergence between Coffea species. The S _H 3-CNL family appeared to evolve following the birth-and-death model, since duplications and deletions were inferred in the evolution of the S _H 3 locus. Gene conversion between paralog members, inter-subgenome sequence exchanges and positive selection appear to be the major forces acting on the evolution of S _H 3-CNL in coffee trees.