High-throughput polymorphism detection and genotyping in <it>Brassica napus</it> using next-generation RAD sequencing

<p>Abstract</p> <p>Background</p> <p>The complex genome of rapeseed (<it>Brassica napus</it>) is not well understood despite the economic importance of the species. Good knowledge of sequence variation is needed for genetics approaches and breeding purposes. We used a diversity set of <it>B. napus</it> representing eight different germplasm types to sequence genome-wide distributed restriction-site associated DNA (RAD) fragments for polymorphism detection and genotyping.</p> <p>Results</p> <p>More than 113,000 RAD clusters with more than 20,000 single nucleotide polymorphisms (SNPs) and 125 insertions/deletions were detected and characterized. About one third of the RAD clusters and polymorphisms mapped to the <it>Brassica rapa</it> reference sequence. An even distribution of RAD clusters and polymorphisms was observed across the <it>B. rapa</it> chromosomes, which suggests that there might be an equal distribution over the <it>Brassica oleracea</it> chromosomes, too. The representation of Gene Ontology (GO) terms for unigenes with RAD clusters and polymorphisms revealed no signature of selection with respect to the distribution of polymorphisms within genes belonging to a specific GO category.</p> <p>Conclusions</p> <p>Considering the decreasing costs for next-generation sequencing, the results of our study suggest that RAD sequencing is not only a simple and cost-effective method for high-density polymorphism detection but also an alternative to SNP genotyping from transcriptome sequencing or SNP arrays, even for species with complex genomes such as <it>B. napus</it>.</p

The effects of quantitative easing on the USA, Japan, Eurozone and Great Britain

This master thesis aims to describe problematics of the effects of unconventional monetary policy, also known as quantitative easing, on economics of USA, Japan, Eurozone and Great Britain, by using empirical analysis of events related to quantitative easing and large BVAR model. In theoretical part of the thesis there are described transmission mechanisms of conventional monetary policy still effective in conditions of interest rates close to zero, as well as channels of unconventional monetary policy. Practical part of the thesis demonstrates analysis of impact of events related to quantitative easing in all the in-scope economics by applying a method of empirical observation of interest rates reactions on every event. Further, based on the received results of the empirical analysis, broader economic effects of quantitative easing are examined by using large BVAR model and afterwards the conclusion is made

Fluorescent amphiphilic heterografted comb polymers comprising biocompatible PLA and PEtOx side chains

A series of amphiphilic heterografted comb polymers comprising various ratios of oligomeric polylactide (PLA) and poly(2-ethyl-2-oxazoline) (PEtOx) side chains was synthesized via the grafting-through method employing the reversible addition-fragmentation chain transfer copolymerization. Two well-defined PLA macromonomers were prepared via ring opening polymerization (ROP) of L-lactide using a calcium-based pre-catalyst, pyrenebutanol as an initiator and methacryloyl chloride as an end-capping agent. The PEtOx macromonomer was obtained from the cationic ROP of EtOx and end-capping with methacrylic acid. The amphiphilic comb polymers self-assembled in aqueous solution to form spherical and wormlike micelles, vesicles and more complex morphologies as a function of the composition, as is evident from dynamic light scattering and cryo-transmission electron microscopy studies. All polymers were found to be non-toxic to L929 cells up to a concentration of 200 mu g mL(-1). Cellular uptake studies with HEK-293 cells by live cell confocal fluorescence microscopy revealed localization in the cytosol after 4 h and suggest an energy-driven cellular uptake mechanism

The Power of Shielding: Low Toxicity and High Transfection Performance of Cationic Graft Copolymers Containing Poly(2-oxazoline) Side Chains

We show the potential of oligo­(2-ethyl-2-oxazoline) (Ox_n)-shielded graft copolymers of (2-aminoethyl)-methacrylate and <i>N</i>-methyl-(2-aminoethyl)-methacrylate for pDNA delivery in HEK cells. For the effect of grafting density and side chain length concerning improved transfection properties through the concept of shielding to be investigated, copolymers were synthesized via the macromonomer method using a combination of cationic ring opening polymerization and reversible addition–fragmentation chain transfer polymerization to vary the degree of grafting (DG = 10 and 30%) as well as the side chain degree of polymerization (DP = 5 and 20). Investigations of the polyplex formation, in vitro flow cytometry, and confocal laser scanning microscopy measurements on the copolymer library revealed classical shielding properties of the Ox side chains, including highly reduced cytotoxicity and a partial decrease in transfection efficiency, as also reported for polyethylene glycol shielding. In terms of the transfection efficiency, the best performing copolymers (<b>A-</b><i><b>g</b></i><b>-Ox</b>_<b>5</b><b>(10)</b> and <b>M-</b><i><b>g</b></i><b>-Ox</b>_<b>5</b><b>(10)</b>) revealed equal or better performances compared to those of the corresponding homopolymers. In particular, the graft copolymers with low DG and side chain DP transfected well with over 10-fold higher IC₅₀ values. In contrast, a DG of 30% resulted in a loss of transfection efficiency due to missing ability for endosomal release, and a side chain DP of 20 hampered the cellular uptake

Beyond Gene Transfection with Methacrylate-Based PolyplexesThe Influence of the Amino Substitution Pattern

Methacrylate-based polymers represent promising nonviral gene delivery vectors, since they offer a large variety of polymer architectures and functionalities, which are beneficial for specific demands in gene delivery. In combination with controlled radical polymerization techniques, such as the reversible addition–fragmentation chain transfer polymerization, the synthesis of well-defined polymers is possible. In this study we prepared a library of defined linear polymers based on (2-aminoethyl)-methacrylate (AEMA), <i>N</i>-methyl-(2-aminoethyl)-methacrylate (MAEMA), and <i>N,N</i>-dimethyl-(2-aminoethyl)-methacrylate (DMAEMA) monomers, bearing pendant primary, secondary, and tertiary amino groups, and investigated the influence of the substitution pattern on their gene delivery capability. The polymers and the corresponding plasmid DNA complexes were investigated regarding their physicochemical characteristics, cytocompatibility, and transfection performance. The nonviral transfection by methacrylate-based polyplexes differs significantly from poly­(ethylene imine)-based polyplexes, as a successful transfection is not affected by the buffer capacity. We observed that polyplexes containing a high content of primary amino groups (AEMA) offered the highest transfection efficiency, whereas polyplexes bearing tertiary amino groups (DMAEMA) exhibited the lowest transfection efficiency. Further insights into the uptake and release mechanisms could be identified by fluorescence and transmission electron microscopy, emphasizing the theory of membrane-pore formation for the time-efficient endosomal release of methacrylate-based vectors