Genetic characterization of a group of commercial African timber species: From genomics to barcoding.

In the last decades, illegal logging has posed a serious threat for the integrity of forest ecosystems and for biodiversity conservation in tropical Africa. Although international treaties and regulatory plans have been implemented to reduce illegal logging, much of the total timber volume is harvested and traded illegally from tropical African forest regions. As a result, the development and the application of analytical tools to enhance the traceability and the identification of wood and related products is critical to enforce international regulations. Among available techniques, DNA barcoding is a promising approach for the molecular identification of plant species. However, although it has been used successfully for the discrimination of animal species, no set of genetic markers is available for the universal identification of plant species. In this work, we firstly characterized the genetic diversity of 17 highly-valuable African timber species from five genera (Afzelia, Guibourtia, Leplea, Milicia, Tieghemella) across their distribution ranges in West and Central Africa using the genome skimming approach in order to reconstruct their chloroplast genomes and nuclear ribosomal DNA. Next, we identified single-nucleotide polymorphisms (SNPs) for the discrimination of closely-related species. In this way, we successfully developed and tested novel species-specific genetic barcodes for species identification.

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

Genome skimming reveals novel plastid markers for the molecular identification of illegally logged African timber species.

Tropical forests represent vast carbon stocks and continue to be key carbon sinks and buffer climate changes. The international policy constructed several mechanisms aiming at conservation and sustainable use of these forests. Illegal logging is an important threat of forests, especially in the tropics. Several laws and regulations have been set up to combat illegal timber trade. Despite significant enforcement efforts of these regulations, illegal logging continues to be a serious problem and impacts for the functioning of the forest ecosystem and global biodiversity in the tropics. Microscopic analysis of wood samples and the use of conventional plant DNA barcodes often do not allow to distinguish closely-related species. The use of novel molecular technologies could make an important contribution for the identification of tree species. In this study, we used high-throughput sequencing technologies and bioinformatics tools to obtain the complete de-novo chloroplast genome of 62 commercial African timber species using the genome skimming method. Then, we performed a comparative genomic analysis that revealed new candidate genetic regions for the discrimination of closely-related species. We concluded that genome skimming is a promising method for the development of plant genetic markers to combat illegal logging activities supporting CITES, FLEGT and the EU Timber Regulation.

A small secreted protein from Zymoseptoria tritici interacts with a wheat E3 ubiquitin ligase to promote disease.

Septoria tritici blotch (STB), caused by the ascomycete fungus Zymoseptoria tritici, is a major threat to wheat production worldwide. The Z. tritici genome encodes many small secreted proteins (ZtSSPs) that are likely to play a key role in the successful colonization of host tissues. However, few of these ZtSSPs have been functionally characterized for their role during infection. In this study, we identified and characterized a small, conserved cysteine-rich secreted effector from Z. tritici which has homologues in other plant pathogens in the Dothideomycetes. ZtSSP2 was expressed throughout Z. tritici infection in wheat, with the highest levels observed early during infection. A yeast two-hybrid assay revealed an interaction between ZtSSP2 and wheat E3 ubiquitin ligase (TaE3UBQ) in yeast, and this was further confirmed in planta using bimolecular fluorescence complementation and co-immunoprecipitation. Down-regulation of this wheat E3 ligase using virus-induced gene silencing increased the susceptibility of wheat to STB. Together, these results suggest that TaE3UBQ is likely to play a role in plant immunity to defend against Z. tritici.

A large-scale species level dated angiosperm phylogeny for evolutionary and ecological analyses.

Phylogenies are a central and indispensable tool for evolutionary and ecological research. Even though most angiosperm families are well investigated from a phylogenetic point of view, there are far less possibilities to carry out large-scale meta-analyses at order level or higher. Here, we reconstructed a large-scale dated phylogeny including nearly 1/8th of all angiosperm species, based on two plastid barcoding genes, matK (incl. trnK) and rbcL. Novel sequences were generated for several species, while the rest of the data were mined from GenBank. The resulting tree was dated using 56 angiosperm fossils as calibration points. The resulting megaphylogeny is one of the largest dated phylogenetic tree of angiosperms yet, consisting of 36,101 sampled species, representing 8,399 genera, 426 families and all orders. This novel framework will be useful for investigating different broad scale research questions in ecological and evolutionary biology.

In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth.

Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.