Diversity of aphyllous Vanilla species in the south-west Indian Ocean region: a challenge for orchid taxonomy, evolution and conservation research

The pantropical genus Vanilla is a member of the Orchidaceae family, one of the largest and most ancient families of angiosperms in the world. It originated in America and differentiated in America, Africa and Asia. About 126 species of Vanilla have been catalogued since the discovery of the genus: 25 can be found in Africa, 31 are indigenous to Asia, New Guinea and Pacific islands, and 70 are distributed in tropical America. The south-west Indian Ocean (SWIO) region, a biodiversity hotspot for orchids, is home to 9 native species of the genus with 7 aphyllous species and 2 leafy species. These 7 aphyllous are represented by two species with yellow flowers (V. humblotii, V. perrieri) and five species with white flowers (V. madagascariensis, V. bosseri, V. decaryana, V. phalaenopsis, V. roscheri). Recent molecular phylogenetic studies showed that these aphyllous species form a recent monophyletic group. Morphological descriptions of these aphyllous species in databases (MNHN, RBG Kew, Tropicos..) and in scientific publications are based on a limited number of individuals. The morphological traits (petals, sepals, label, and stems) are very similar between species and do not provide reliable identification despite possible flower size differences. Moreover, the species have more or less the same flowering period (October-December) and their geographical distribution areas overlap in Madagascar, increasing the probability of finding some sympatric species such as V. madagascariensis, V. bosseri and V. perrieri, and therefore possible hybrids. As the available information on genetic structuring and phylogeny of these species is insufficient, the resolution of their taxonomy is problematic. Aphyllous Vanilla species from the SWIO islands are thus an excellent model for understanding the evolution of orchids. For their conservation and to confirm the identity of these aphyllous species, an integrated approach with classical taxonomy using a large number of samples, intense fieldwork on biology and ecology, and molecular studies using variable markers is currently underway

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

Background: Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. Results: An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. Conclusions: This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.The research was financially supported by the Academy of Finland Centre of Excellence (#307335), NordForsk Nordic Centre of Excellence (#82845) and Jane and Aatos Erkko Foundation (#4605–26422). The work also received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Action—Innovative Training Network 2017 (#764920), and Fundação para a Ciência e a Tecnologia (CEECIND/00259/2017 to CCP)