A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors

Background

The diploid, Solanum caripense, a wild relative of potato and tomato, possesses valuable resistance to potato late blight and we are interested in the genetic base of this resistance. Due to extremely low levels of genetic variation within the S. caripense genome it proved impossible to generate a dense genetic map and to assign individual Solanum chromosomes through the use of conventional chromosome-specific SSR, RFLP, AFLP, as well as gene- or locus-specific markers. The ease of detection of DNA polymorphisms depends on both frequency and form of sequence variation. The narrow genetic background of close relatives and inbreds complicates the detection of persisting, reduced polymorphism and is a challenge to the development of reliable molecular markers. Nonetheless, monomorphic DNA fragments representing not directly usable conventional markers can contain considerable variation at the level of single nucleotide polymorphisms (SNPs). This can be used for the design of allele-specific molecular markers. The reproducible detection of allele-specific markers based on SNPs has been a technical challenge.

Results

We present a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD) markers. These markers proved highly efficient for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA) base. We demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII) markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles of 16 chromosome-specific COSII markers and to assign eight of the twelve linkage groups to consensus Solanum chromosomes.

Conclusion

The method based on individual allelic variants allows for a level-of-magnitude higher resolution of genetic variation than conventional marker techniques. We show that the majority of monomorphic molecular marker fragments from organisms with reduced heterozygosity levels still contain SNPs that are sufficient to trace individual alleles.     

A broad spectrum screening of Schmallenberg virus antibodies in wildlife animals in Germany

To identify native wildlife species possibly susceptible to infection with Schmallenberg virus (SBV), a midge-transmitted orthobunyavirus that predominantly infects domestic ruminants, samples from various free-living ruminants, but also carnivores, small mammals and wild boar were analyzed serologically. Before 2011, no SBV-specific antibodies were detectable in any of the tested species, thereafter, a large proportion of the ruminant population became seropositive, while every sample taken from carnivores or small mammals tested negative. Surprisingly, SBV-specific-antibodies were also present in a large number of blood samples from wild boar during the 2011/2012 and 2012/2013 hunting seasons. Hence, free-ranging artiodactyls may play a role as wildlife host.     

Spontaneous outcrossing in tomato depends on cultivar and environment and varies between individual flowers

Knowledge about the degree of spontaneous outcrossing of diverse genotypes is essential for breeding programmes, maintenance breeding, and seed production. For tomato (Lycopersicon esculentum Mill.), very limited scientific evidence for genotypic differences is available and evidence from Europe is scarce. To close this knowledge gap, six cultivars were investigated in three Central European locations as part of the Organic Outdoor Tomato Project. To determine outcrossing rates, the monogenetic “cut‐leaf” trait, which is dominant over the “potato‐leaf” trait, was used as morphological marker. The observed range of outcrossing was 0.0%–5.2%. Outcrossing was significantly influenced by cultivar and environment. The outcrossing rate of individual flowers varied within cultivars ranging from 0% to 37%. The potential of newly opened flowers to accept foreign pollen varied largely with the cultivar. Genotypic differences could partly be linked to flower morphology traits. The potential for recombination between tomato genotypes is generally very low but can be a source for new variation in on‐farm management.     

The Use of Positron Emission Tomography in Soft Tissue Sarcoma Patients under Therapy with Trabectedin

Background

We used 2-deoxy-2-[¹⁸F] fluoro-D-glucose (FDG) positron emission tomography (PET) to evaluate the FDG uptake in patients with advanced and/or metastatic soft tissue sarcoma (STS) undergoing therapy with Ecteinascidin-743 (ET-743, Trabectedin, Yondelis™).

Patients and Methods

The pilot study included nine patients with metastatic STS receiving a minimum of one cycle of treatment with trabectedin. Patients were examined using PET prior to onset of therapy and after completion of one or three cycles of trabectedin. Restaging according to Response Evaluation Criteria in Solid Tumours (RECIST) was performed in parallel using computed tomography (CT) and/or magnetic resonance imaging (MRI) and served for reference.

Results

Clinical outcome of nine evaluable patients was as follows: one patient with partial remission (PR), three patients with stable disease (SD), and five patients with progressive disease (PD). A more than 40% decrease of the standardized uptake value (SUV) of sequential PET examination could be demonstrated for the responding patient (PR), whereas patients with SD or PD showed a stable SUV, but no increase in SUV.

Conclusion

To our knowledge, this is the first small series of patients being treated with trabectedin and monitored using sequential PET imaging demonstrating SUV stabilization in nearly all monitored patients.     

Molecular characterization of PRR13 and its tissue-specific expression in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

The proline-rich protein 13 (PRR13) is reported to be a key regulator of the resistance to cytostatica by decreasing the copy number of the proapoptotic gene thrombospondin-1. We isolated and characterized the complete PRR13 gene sequence of rainbow trout (Oncorhynchus mykiss). The gene comprises four exons and three introns, the latter of comparatively short lengths (100–811 bp). The full-length PRR13 cDNA consists of 1,101 nucleotides, including an open reading frame of 563 bp, which is predicted to encode a 187 amino acid protein with a molecular mass of 18.8 kDa. A continuous stretch of ten serine residues at the C-terminus is highly conserved and characteristic for vertebrate PRR13, but not for other known proline-rich proteins. Phylogenetic analyses suggest a clear separation of teleostean PRR13 proteins and those from mammalian and reptilian species. Comparison of the tissue-specific PRR13 mRNA abundance in two strains of the rainbow trout coastal form (TCO Steelhead II-WA vs. BORN Steelhead II-Germany) revealed an increased expression in the BORN trout in nearly all examined tissues. The major expression differences were detected in gill (2.29-fold) and in liver tissue (2.16-fold). Hence, the increased PRR13 expression in BORN trout might cause improved protection from natural cytostatica and therefore support our assumption that PRR13 is a candidate gene possibly involved in the varying ability of the two rainbow trout strains to handle environmental stress under local conditions of the Southern Baltic.     

Effect of extreme acid and alkali treatment on surface properties of soils

Effects of natural or anthropogenic soil acidification and alkalization on chemical or biological properties have been studied extensively while little is known about changes in physicochemical characteristics, such as surface area or adsorption energy. To investigate this, samples of six Polish soils (Inceptisols, Mollisols) and three Korean soils (Alfisols, Ultisols) of different origin and mineral composition were acidified and alkalized with elevated concentrations of hydrochloric acid and sodium hydroxide ranging from 0.001 to 1 mol dm^—3. Surface properties of these soils were studied. The surface area and average adsorption energy decreased in general in both treatments. The treatments induced the decrease in amount of high and medium energy centers, however the fraction of low energy centers increased. The behavior of surface properties differed from the above at treatments at highest reagent concentrations, especially for Korean soils, rich in clay and iron oxides. The general pattern of the adsorption energy decrease observed in most of the soils indicates that the overall water binding forces become lower after treatments. In this case the soil water may be more available for plants, despite its amount decreases.