Romain Guyot

Rapid Genome Divergence at Orthologous Low Molecular Weight Glutenin Loci of the A and Am Genomes of Wheat

To study genome evolution in wheat, we have sequenced and compared two large physical contigs of 285 and 142 kb covering orthologous low molecular weight (LMW) glutenin loci on chromosome 1AS of a diploid wheat species (Triticum monococcum subsp monococcum) and a tetraploid wheat species (Triticum turgidum subsp durum). Sequence conservation between the two species was restricted to small regions containing the orthologous LMW glutenin genes, whereas >90% of the compared sequences were not conserved. Dramatic sequence rearrangements occurred in the regions rich in repetitive elements. Dating of long terminal repeat retrotransposon insertions revealed different insertion events occurring during the last 5.5 million years in both species. These insertions are partially responsible for the lack of homology between the intergenic regions. In addition, the gene space was conserved only partially, because different predicted genes were identified on both contigs. Duplications and deletions of large fragments that might be attributable to illegitimate recombination also have contributed to the differentiation of this region in both species. The striking differences in the intergenic landscape between the A and Am genomes that diverged 1 to 3 million years ago provide evidence for a dynamic and rapid genome evolution in wheat species.

CACTA Transposons in Triticeae. A Diverse Family of High-Copy Repetitive Elements

In comparison with retrotransposons, which comprise the majority of the Triticeae genomes, very few class 2 transposons have been described in these genomes. Based on the recent discovery of a local accumulation of CACTA elements at theGlu-A3 loci in the two wheat species Triticum monococcum and Triticum durum, we performed a database search for additional such elements in Triticeae spp. A combination of BLAST search and dot-plot analysis of publicly available Triticeae sequences led to the identification of 41 CACTA elements. Only seven of them encode a protein similar to known transposases, whereas the other 34 are considered to be deletion derivatives. A detailed characterization of the identified elements allowed a further classification into seven subgroups. The major subgroup, designated the “Caspar ” family, was shown by hybridization to be present in at least 3,000 copies in theT. monococcum genome. The close association of numerous CACTA elements with genes and the identification of several similar elements in sorghum (Sorghum bicolor) and rice (Oryza sativa) led to the conclusion that CACTA elements contribute significantly to genome size and to organization and evolution of grass genomes.

Whole-Genome Comparison of Leucine-Rich Repeat Extensins in Arabidopsis and Rice. A Conserved Family of Cell Wall Proteins Form a Vegetative and a Reproductive Clade

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. TheLRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into “reproductive” and “vegetative” LRXs. The four Arabidopsis genes of the “reproductive” clade are specifically expressed in pollen, whereas the seven “vegetative” genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum)LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.

Plant dicistronic tRNA-snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z.

Small nucleolar RNAs (snoRNAs) guiding modifications of ribosomal RNAs and other RNAs display diverse modes of gene organization and expression depending on the eukaryotic system: in animals most are intron encoded, in yeast many are monocistronic genes and in plants most are polycistronic (independent or intronic) genes. Here we report an unprecedented organization: plant dicistronic tRNA–snoRNA genes. In Arabidopsis thaliana we identified a gene family encoding 12 novel box C/D snoRNAs (snoR43) located just downstream from tRNAGly genes. We confirmed that they are transcribed, probably from the tRNA gene promoter, producing dicistronic tRNAGly–snoR43 precursors. Using transgenic lines expressing a tagged tRNA–snoR43.1 gene we show that the dicistronic precursor is accurately processed to both snoR43.1 and tRNAGly. In addition, we show that a recombinant RNase Z, the plant tRNA 3′ processing enzyme, efficiently cleaves the dicistronic precursor in vitro releasing the snoR43.1 from the tRNAGly. Finally, we describe a similar case in rice implicating a tRNAMet‐e expressed in fusion with a novel C/D snoRNA, showing that this mode of snoRNA expression is found in distant plant species.

Complex organization and evolution of the tomato pericentromeric region at the FER gene locus.

Tomato (Lycopersicon esculentum) is a model species for molecular biology research and a candidate for large-scale genome sequencing. Pericentromeric heterochromatin constitutes a large portion of the tomato chromosomes. However, the knowledge of the structure, organization, and evolution of such regions remains very limited. Here, we report the analysis of a 198-kb sequence near the FER gene, located in a distal part of pericentromeric heterochromatin on the long arm of tomato chromosome 6. Nine genes, one pseudogene, and 55 transposable elements (TEs) were identified, showing a low gene density (19.8 kb/gene) and a high content of transposable elements (>45% of the sequence). Six genes (56B23_g3, g5, g7, g8, g9, and g10) have perfect matches (>98% identity) with tomato expressed sequence tags. Two genes (56B23_g1 and g6), which share <98% sequence identity with expressed sequence tags, were confirmed for transcriptional activity by reverse transcription-PCR. The genes were not uniformly distributed along the sequence and grouped into gene islands separated by stretches of retrotransposons, forming a pattern similar to that found in the gene-rich regions of the large genomes of maize (Zea mays) and Triticeae. Long terminal repeat retrotransposons account for 60% of the TE sequence length. Sixteen of 55 TEs were completely new and remain unclassified. Surprisingly, five of the seven identified DNA transposons were closely associated with coding regions. The action of transposable elements and DNA rearrangements form the molecular basis of the dynamic genome evolution at the FER locus. Multiple rounds of genome duplication in Arabidopsis (Arabidopsis thaliana) and subsequent gene loss have generated a mosaic pattern of conservation between tomato and Arabidopsis orthologous sequences. Our data show that the distal parts of pericentromeric heterochromatin may contain many valuable genes and that these regions form an evolutionary active part of the tomato genome.

Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae)

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1-copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution.

A new structural element containing glycine-rich proteins and rhamnogalacturonan I in the protoxylem of seed plants

The water pipes of elongating plant organs are the result of programmed cell death and are formed by the walls of dead and empty protoxylem elements. These protoxylem elements are passively elongated many times by the surrounding tissue before they are replaced and collapse. Well-known adaptations for this unique task include the characteristic secondary wall thickenings, forming rings and helices. A new, clearly distinct structural element containing glycine-rich proteins is now visualized for the first time, using confocal laser scanning microscopy in the mature protoxylem of elongating organs of seed plants. This structural element is arranged along the longitudinal axis of the protoxylem elements. It interconnects the secondary wall thickenings within and between protoxylem elements, as well as the protoxylem with other cell types such as xylem parenchyma cells and metaxylem elements. The structural element is stable against detergent extractions, proteinase, pectinase and cellulase hydrolysis, and is closely associated with rhamnogalacturonan-I, a pectic polysaccharide. The results clearly demonstrate that the cell wall of protoxylem cells is a highly dynamic and complex structure. The typical polysaccharide-rich primary wall of living and elongating plant cells is progressively modified and finally replaced by a protein-rich wall in the dead and passively stretched protoxylem elements. These glycine-rich walls originated early in the evolution of the seed plants as confirmed by the analysis of genomic information.

Genotyping-by-sequencing provides the first well-resolved phylogeny for coffee (Coffea) and insights into the evolution of caffeine content in its species: GBS coffee phylogeny and the evolution of caffeine content

A comprehensive and meaningful phylogenetic hypothesis for the commercially important coffee genus (Coffea) has long been a key objective for coffee researchers. For molecular studies, progress has been limited by low levels of sequence divergence, leading to insufficient topological resolution and statistical support in phylogenetic trees, particularly for the major lineages and for the numerous species occurring in Madagascar. We report here the first almost fully resolved, broadly sampled phylogenetic hypothesis for coffee, the result of combining genotyping-by-sequencing (GBS) technology with a newly developed, lab-based workflow to integrate short read next-generation sequencing for low numbers of additional samples. Biogeographic patterns indicate either Africa or Asia (or possibly the Arabian Peninsula) as the most likely ancestral locality for the origin of the coffee genus, with independent radiations across Africa, Asia, and the Western Indian Ocean Islands (including Madagascar and Mauritius). The evolution of caffeine, an important trait for commerce and society, was evaluated in light of our phylogeny. High and consistent caffeine content is found only in species from the equatorial, fully humid environments of West and Central Africa, possibly as an adaptive response to increased levels of pest predation. Moderate caffeine production, however, evolved at least one additional time recently (between 2 and 4Mya) in a Madagascan lineage, which suggests that either the biosynthetic pathway was already in place during the early evolutionary history of coffee, or that caffeine synthesis within the genus is subject to convergent evolution, as is also the case for caffeine synthesis in coffee versus tea and chocolate.

Genetic structure and diversity of coffee (Coffea) across Africa and the Indian Ocean islands revealed using microsatellites

BACKGROUND AND AIMS The coffee genus (Coffea) comprises 124 species, and is indigenous to the Old World Tropics. Due to its immense economic importance, Coffea has been the focus of numerous genetic diversity studies, but despite this effort it remains insufficiently studied. In this study the genetic diversity and genetic structure of Coffea across Africa and the Indian Ocean islands is investigated. METHODS Genetic data were produced using 13 polymorphic nuclear microsatellite markers (simple sequence repeats, SSRs), including seven expressed sequence tag-SSRs, and the data were analysed using model- and non-model-based methods. The study includes a total of 728 individuals from 60 species. KEY RESULTS Across Africa and the Indian Ocean islands Coffea comprises a closely related group of species with an overall pattern of genotypes running from west to east. Genetic structure was identified in accordance with pre-determined geographical regions and phylogenetic groups. There is a good relationship between morpho-taxonomic species delimitations and genetic units. Genetic diversity in African and Indian Ocean Coffea is high in terms of number of alleles detected, and Madagascar appears to represent a place of significant diversification in terms of allelic richness and species diversity. CONCLUSIONS Cross-species SSR transferability in African and Indian Ocean islands Coffea was very efficient. On the basis of the number of private alleles, diversification in East Africa and the Indian Ocean islands appears to be more recent than in West and West-Central Africa, although this general trend is complicated in Africa by the position of species belonging to lineages connecting the main geographical regions. The general pattern of phylogeography is not in agreement with an overall east to west (Mascarene, Madagascar, East Africa, West Africa) increase in genome size, the high proportion of shared alleles between the four regions or the high numbers of exclusive shared alleles between pairs or triplets of regions.

Large distribution and high sequence identity of a Copia-type retrotransposon in angiosperm families

Retrotransposons are the main component of plant genomes. Recent studies have revealed the complexity of their evolutionary dynamics. Here, we have identified Copia25 in Coffea canephora, a new plant retrotransposon belonging to the Ty1-Copia superfamily. In the Coffea genomes analyzed, Copia25 is present in relatively low copy numbers and transcribed. Similarity sequence searches and PCR analyses show that this retrotransposon with LTRs (Long Terminal Repeats) is widely distributed among the Rubiaceae family and that it is also present in other distantly related species belonging to Asterids, Rosids and monocots. A particular situation is the high sequence identity found between the Copia25 sequences of Musa, a monocot, and Ixora, a dicot species (Rubiaceae). Our results reveal the complexity of the evolutionary dynamics of the ancient element Copia25 in angiosperm, involving several processes including sequence conservation, rapid turnover, stochastic losses and horizontal transfer.