Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations

Background  

The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal.     

Effect of Leptin on In Vivo Goat Embryo Production

The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133 mg follicle‐stimulating hormone (FSH) i.m. in six descending doses at 12‐h intervals. The goats received 4.8 μg/kg human recombinant leptin s.c. (leptin group, n = 5) or phosphate‐buffered saline (PBS) (control group, n = 5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6 days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick‐end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E₂) and progesterone (P₄) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p < 0.005), fewer embryos classified as degenerated (p < 0.001) and fewer TUNEL‐positive cells/blastocyst (p < 0.001). The number of transferable embryos was positively correlated with E₂ concentrations on day of oestrus (r = 0.562; p < 0.01) and P₄ concentrations on the day of embryo collection (r = 0.912; p < 0.001). We concluded that in vivo leptin administration during FSH treatment improved embryo quality and affected ovarian steroidogenesis in superovulated goats.     

Pathogens as drivers of population declines: The importance of systematic monitoring in great apes and other threatened mammals

Until recently, the focus of great ape behavioural and ecological research has been distinct from the focus of scientists working in medical and veterinary sciences. More scientists are calling for a connection between medical and field research due to recent disease outbreaks in great apes, including Ebola, and indications of cross-transmission of Ebola and other viruses between primates and humans. A major limitation to progress is the lack of information on infectious diseases and their transmission in wild primates. Here, we present examples of successful pathogen detection in wild great apes and describe approaches and techniques that can be used in the field, focusing in particular on investigation of deaths and non-invasive sample collection. This interdisciplinary approach is providing new insights to infectious diseases of great apes and is helping to protect the health of great ape populations. This framework can also be applied to other mammals under threat from infectious diseases, including African wild dogs, seals and Tasmanian devils. In addition to providing benefits for great ape conservation, research that integrates infectious disease with primate ecology provides insights to emerging diseases in humans and the role of disease in primate evolution.     

Protein-DNA interactions in vivo upstream of a cell cycle-regulated human H4 histone gene

Cell cycle-dependent histone genes are transcribed at a basal level throughout the cell cycle, with a three- to fivefold increase during early S phase. Protein-DNA interactions in the 5' promoter region of a cell cycle-regulated human H4 histone gene have been analyzed at single-nucleotide resolution in vivo. This region contains two sites, with four potential protein-binding domains, at which the DNA is protected from reaction with dimethyl sulfate in cells and from digestion with deoxyribonuclease I in nuclei. These protein-DNA interactions persist during all phases of the cell cycle and dissociate with 0.16 to 0.2M sodium chloride.     

High throughput phenotyping for aphid resistance in large plant collections

ABSTRACT: BACKGROUND: Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. RESULTS: In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. CONCLUSIONS: This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.