High-energy x-ray Talbot-Lau radiography of a human knee.

We report on a radiographic measurement of an ex vivo human knee using a grating-based phase-contrast imaging setup and a medical x-ray tube at a tube voltage of 70 kV. The measurement has been carried out using a Talbot-Lau setup that is suitable to achieve a high visibility in the energy regime of medical imaging. In a medical reading by an experienced trauma surgeon signatures of chondrocalcinosis in the medial meniscus have been identified more evidently using the dark-field image in comparison to the conventional attenuation image. The analysis has been carried out at various dose levels down to 0.14 mGy measured as air kerma, which is a dose comparable to clinically used radiographic devices. The diagnosis has been confirmed by a histological analysis of the meniscus tissue. In the introduced high-frequency filtered phase-contrast image the anterior and posterior horn of the medial meniscus and the posterior cruciate ligament have also been visible. Furthermore, atherosclerotic plaque is visible in both imaging modalities, attenuation and dark-field, despite the presence of overlaying bone. This measurement, for the first time, proves the feasibility of Talbot-Lau x-ray imaging at high-energy spectra above 40 kVp and reasonable dose levels with regard to spacious and dense objects.

Optimisation of image reconstruction for phase-contrast x-ray Talbot-Lau imaging with regard to mechanical robustness.

X-ray grating-based phase-contrast imaging opens new opportunities, inter alia, in medical imaging and non-destructive testing. Because, information about the attenuation properties and about the refractive properties of an object are gained simultaneously. Talbot-Lau imaging requires the knowledge of a reference or free-field image. The long-term stability of a Talbot-Lau interferometer is related to the time span of the validity of a measured reference image. It would be desirable to keep the validity of the reference image for a day or longer to improve feasibility of Talbot-Lau imaging. However, for example thermal and other long-term external influences result in drifting effects of the phase images. Therefore, phases are shifting over time and the reference image is not valid for long-term measurements. Thus, artifacts occur in differential phase-contrast images. We developed an algorithm to determine the differential phase-contrast image with the help of just one calibration image, which is valid for a long time-period. With the help of this algorithm, called phase-plane-fit method, it is possible to save measurement-time, as it is not necessary to take a reference image for each measurement. Additionally, transferring the interferometer technique from laboratory setups to conventional imaging systems the necessary rigidity of the system is difficult to achieve. Therefore, short-term effects like vibrations or distortions of the system lead to imperfections within the phase-stepping procedure. Consequently, artifacts occur in all three image modalities (differential phase-contrast image, attenuation image and dark-field image) of Talbot-Lau imaging. This is a problem with regard to the intended use of phase-contrast imaging for example in clinical routine or non-destructive testing. In this publication an algorithm of Vargas et al is applied and complemented to correct inaccurate phase-step positions with the help of a principal component analysis (PCA). Thus, it is possible to calculate the artifact free images. Subsequently, the whole algorithm is called PCA minimization algorithm.

Hierarchical classification of switchgrass genotypes using SSR and chloroplast sequences: ecotypes, ploidies, gene pools, and cultivars.

Switchgrass (Panicum virgatum L.) is an important crop for bioenergy feedstock development. Switchgrass has two main ecotypes: the lowland ecotype being exclusively tetraploid (2n = 4x = 36) and the upland ecotype being mainly tetraploid and octaploid (2n = 8x = 72). Because there is a significant difference in ploidy, morphology, growth pattern, and zone of adaptation between and within the upland and lowland ecotypes, it is important to discriminate switchgrass plants belonging to different genetic pools. We used 55 simple sequence repeats (SSR) loci and six chloroplast sequences to identify patterns of variation between and within 18 switchgrass cultivars representing seven lowland and 11 upland cultivars from different geographic regions and of varying ploidy levels. We report consistent discrimination of switchgrass cultivars into ecotype membership and demonstrate unambiguous molecular differentiation among switchgrass ploidy levels using genetic markers. Also, SSR and chloroplast markers identified genetic pools related to the geographic origin of the 18 cultivars with respect to ecotype, ploidy, and geographical, and cultivar sources. SSR loci were highly informative for cultivar fingerprinting and to classify plants of unknown origin. This classification system is the first step toward developing switchgrass complementary gene pools that can be expected to provide a significant heterotic increase in biomass yield.

Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation.

A cytosine DNA methyltransferase containing a chromodomain, Zea methyltransferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal motifs characteristic of eukaryotic and prokaryotic DNA methyltransferases. We used a reverse genetics approach to determine the function of the Zmet2 gene. Plants homozygous for a Mutator transposable element insertion into motif IX had a 13% reduction in methylated cytosines. DNA gel blot analysis of these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at CpNpG sites. No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants. Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences.

Epigenetic aspects of somaclonal variation in plants.

Somaclonal variation is manifested as cytological abnormalities, frequent qualitative and quantitative phenotypic mutation, sequence change, and gene activation and silencing. Activation of quiescent transposable elements and retrotransposons indicate that epigenetic changes occur through the culture process. Epigenetic activation of DNA elements further suggests that epigenetic changes may also be involved in cytogenetic instability through modification of heterochromatin, and as a basis of phenotypic variation through the modulation of gene function. The observation that DNA methylation patterns are highly variable among regenerated plants and their progeny provides evidence that DNA modifications are less stable in culture than in seed-grown plants. Future research will determine the relative importance of epigenetic versus sequence or chromosome variation in conditioning somaclonal variation in plants.

Conserved plant genes with similarity to mammalian de novo DNA methyltransferases.

DNA methylation plays a critical role in controlling states of gene activity in most eukaryotic organisms, and it is essential for proper growth and development. Patterns of methylation are established by de novo methyltransferases and maintained by maintenance methyltransferase activities. The Dnmt3 family of de novo DNA methyltransferases has recently been characterized in animals. Here we describe DNA methyltransferase genes from both Arabidopsis and maize that show a high level of sequence similarity to Dnmt3, suggesting that they encode plant de novo methyltransferases. Relative to all known eukaryotic methyltransferases, these plant proteins contain a novel arrangement of the motifs required for DNA methyltransferase catalytic activity. The N termini of these methyltransferases contain a series of ubiquitin-associated (UBA) domains. UBA domains are found in several ubiquitin pathway proteins and in DNA repair enzymes such as Rad23, and they may be involved in ubiquitin binding. The presence of UBA domains provides a possible link between DNA methylation and ubiquitin/proteasome pathways.

Genetic Variability Among Maize Inbred Lines for Resistance to the High Plains Virus-Wheat Streak Mosaic Virus Complex.

High plains virus (HPV) is a pathogen that causes a severe disease, especially in susceptible corn genotypes. The virus is transmitted by the eriophyid mite Aceria tosichella, which also transmits wheat streak mosaic virus (WSMV). This often results in a mixed infection by these two viruses. Genetic variability for resistance to the HPV exists among maize inbred lines but has not been characterized. The disease reaction of 30 maize inbred lines to the mixed infection and to WSMV alone was characterized in this study. Evaluation was based on symptom expression and virus titer (HPV and WSMV), as measured by enzyme-linked immunosorbent assay. All lines showed some HPV symptoms, which ranged from a few visible spots to rapid plant death. HPV-resistant inbreds include B73 and B14. Susceptible inbreds include W64A, Wf9, H100, N213, N215, and N194. Five of the six inbreds most severely affected by HPV also had a high WSMV titer, although they displayed few symptoms when inoculated with WSMV alone. Inbred N194 was one of the genotypes most susceptible to HPV, but it showed no detectable WSMV titer. Seedlings of highly susceptible genotypes often died within 2 weeks of infection.

Root tip cell cycle synchronization and metaphase-chromosome isolation suitable for flow sorting in common wheat (Triticum aestivum L.).

An efficient procedure for cell-cycle synchronization in meristematic root tips was achieved in common wheat. Treatment parameters for synchronizing the cell cycle of root tip meristem cells, such as time-course and applied concentrations of various chemicals, were systematically tested and optimized by flow cytometric analysis of isolated nuclei. High mitotic indices (69.5% in the root tip meristematic area) were routinely obtained by treating germinating seeds with 1.25 mM hydroxyurea for 16 h, followed by incubation in a hydroxyurea-free solution for 2 h, and treatment with 1 μM trifluralin for 4 h. Uniform seed germination prior to treatment is very important for achieving consistently high metaphase indices in the root tips. Large numbers of metaphase chromosomes, suitable for flow cytometric analysis and sorting, were isolated from synchronized root tip cells. Flow sorted wheat chromosomes, via univariate and bivariate analysis, showed four major chromosome peaks. Each discrete peak may represent wheat chromosome types with similar DNA content. Bivariate flow karyotyping based on AT and GC content did not improve the separation of wheat chromosomes.

Cell synchronization and isolation of metaphase chromosomes from maize (Zea mays L.) root tips for flow cytometric analysis and sorting.

Accumulation of cells containing metaphase chromosomes is an important step in cytological analyses and chromosome sorting procedures. The goal of this research was to optimize treatment parameters to synchronize the cell cycle of maize root tip meristem cells. Levels of hydroxyurea, a DNA synthesis inhibitor, were assessed for their utility in accumulating cells at the G1 phase of the cell cycle. Trifluralin, amiprophos-methyl, and colchicine were used to accumulate cells containing metaphase chromosomes upon release from hydroxyurea inhibition. Optimal mitotic indices were achieved by treating seedlings with 5 mM hydroxyurea for 18 h, incubating for 1 h without chemical treatment to release the hydroxyurea block, and then treating emerging roots with 1 μM trifluralin for 4 h. The mitotic index of synchronized maize root tips was over 70%. Uniformity of synchronization depended upon selection of seeds with emerging radicles that were similar in length at the time of treatment. Suspensions of intact chromosomes were prepared by a simple slicing procedure. The chromosome preparations were found to be suitable for flow cytometric characterization and sorting. Chromosome peaks of the observed flow karyotype resembled the predicted flow karyotype calculated on the basis of maize chromosome size. Key words : flow karyotype, hydroxyurea, plant chromosome sorting, trifluralin.

A PCR assay for detection of a 2RL.2BS wheat-rye chromosome translocation.

A 2RL.2BS wheat-rye translocation, present in the wheat germplasm line Hamlet, carries a gene for resistance to Hessian fly biotype L, one of the most virulent biotypes presently encountered in wheat production environments. Unlike several other wheat-rye chromosome translocations common in wheat breeding programs, 2RL lacks genes encoding storage proteins or other easily selected markers. Oligonucleotide primers synthesized from published sequences derived from the R173 family of moderately repetitive rye DNA were used in the DNA polymerase chain reaction (PCR) to identify specific markers for 2RL. The same primers, when used with DNA extracted from additional wheat-rye translocation lines of importance to the wheat breeding community, gave distinctive PCR products for each genotype. The single primer pair, PAWS5 and PAWS6, may, therefore, have wide applicability for the identification of wheat-rye chromosomal translocations presently encountered in wheat breeding populations.

Genetic instability of plant tissue cultures: breakdown of normal controls.

Plants regenerated from relatively undifferentiated callus cultures possess a vast array of genetic changes. Such variations can result in useful agricultural and horticultural products. For other purposes, however, variations in traits other than those of interest may be undesirable--for example, using cultured cells for genetic engineering. Any steps made toward understanding the basis of tissue culture-induced genetic variation should be helpful in developing a more stable and manipulatable somatic cell system. This review provides a glimpse at the specific kinds of genetic changes encountered among regenerated plants and their progeny. Included among these variations are cytosine methylation alterations of the genome. The repeat-induced point mutation (RIP) phenomenon, reported for filamentous fungi, is invoked to provide a framework to consider the origin of variation in plant tissue cultures.

Tissue culture-induced DNA methylation variation in maize.

Twenty-one progeny lines derived from tissue cultures of two embryo sources of maize inbred strain A188 were examined for DNA methylation changes. Total DNA was cut with the isoschizomers Hpa II and Msp I and probed with 18 single-copy Pst I genomic clones and two cDNA clones. Eight of these probes could detect both increases and decreases in methylation. With these probes 39% of the families were found to contain an altered methylation pattern. All changes represented a decrease in methylation. The other 12 probes could detect only increases in methylation; no methylation variation was seen with these probes. Fifteen percent of the methylation changes were homozygous in the original regenerated plant. Changes were stably inherited upon two generations of self-pollination. No sequence variation was observed in Msp I-digested DNA from the same 21 progeny lines. Certain probes detected methylation changes much more often than others. Our study provides evidence that demethylation occurs at a high frequency and could be an important cause of tissue culture-induced variation. Occurrence of the frequent homozygous alterations in original regenerated plants implies a non-random mutational mechanism.

Use of near-isogenic lines derived by backcrossing or selfing to map qualitative traits.

Near-isogenic lines (NILs) are a valuable resource for detecting linkages between qualitative trait loci and molecular markers. Molecular marker studies are expensive and methods that require genotyping fewer individuals, such as the NIL-analysis method, are desirable. We present a theory for using sets of NILs to detect linkages between molecular markers and introgressed loci. The probability that a marker a specific distance from the introgressed gene will have a donor parent allele in a near-isogenic line is a function of the distance between the marker and the gene, and the number of back-crosses and/or selfs used in deriving the NIL. The binomial probability formula is used to calculate the probability of having a donor parent allele at a given marker when sets of NILs are used. The formulae given allow calculation of the probability that a marker is linked to the introgressed gene, as well as the probability that a gene will be successfully detected when using given numbers of NILs, backcrosses, and molecular markers.