Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

<p>Abstract</p> <p>Background</p> <p>A recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, <it>Peronosclerospora sorghi</it>. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.</p> <p>Results</p> <p>Among the 55 primers pairs designed from clones from pathotype 3 of <it>P. sorghi</it>, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from <it>P. sorghi</it>, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (<it>P. maydis & P. philippinensis</it>), sugar cane (<it>P. sacchari</it>), pearl millet (<it>Sclerospora graminicola</it>) and rose (<it>Peronospora sparsa</it>) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to <it>P. philippinensis </it>(one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 <it>Peronosclerospora, Peronospora and Sclerospora </it>spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of <it>Peronosclerospora sorghi </it>in cluster I, five isolates of <it>P. maydis </it>and three isolates of <it>P. sacchari </it>in cluster II and five isolates of <it>Sclerospora graminicola </it>in cluster III).</p> <p>Conclusion</p> <p>To our knowledge, this is the first attempt to extensively develop SSR markers from <it>Peronosclerospora </it>genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.</p

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion

Isolation and characterization of five ozone-inductible cDNA clones from Atriplax canescens (saltbush)

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to [email protected], referencing the URI of the item.Includes bibliographical references.Prolonged exposure of plants to ozone inhibits growth and development and produces visible signs of damage. Within a few hours of exposure, certain biochemical alterations have been observed with the induction of several stress-related proteins. accumulation of several polya RNAS. CDNA clones derived from five different polya RNAs which accumulate in ozone-stressed shrub leaves were isolated by differential screening methods. Alteration in gene expression which occurs with ozone, sulfur dioxide and water deficit were examined, and those cDNAs were sequenced. Clone 012-1, hybridized with three RNA species, is initially induced in response to both ozone and water deficit stress, but is repressed to prolonged ozone and sulfur dioxide exposure. Clone 012-1 has strong homology with small subunit RRNA. Northern blots suggest that transcripts hybridized to 012-1 are a naked species (about 1.45 kb) and are subfragments (1.0 and 0.45 kb) of 1.4 kb species. 0112A-3 showed an increase in response to two pollutants, but not to water deficit. 0112A-3 encodes a polypeptide which has homology with wound-inducible protein inhibitors. MRNA species corresponding to 018-3 are transiently induced in response to sulfur dioxide and water deficit stress, and are late-induced by ozone stress. The 018-3 protein is Ozone fumigation to Atriplex canescens (saltbush) caused the homologous to thiol proteases. Clone OID2-2 and OIS14-3 show similar patterns in gene expression Transcript levels of the two genes rise rapidly and remain at a high level by seven days after ozone fumigation, and rise steadily in response to water deficit and sulfur dioxide stress. The deduced amino acids of both clones are glycine-rich proteins, and they have repeated motifs, (G-G-G-Y-G-H)n, and putative cell wall-targeting signal peptides. Northern analyses indicate that plant cells respond to ozone stress by increasing transcription of several genes. Furthermore, the expression of ozone-inducible genes varies with respect to the time course of induction and different stresses such as water-deficit and sulfur dioxide stress. The encoded proteins suggest that: first, ozone stress may generate wounding damage to increase the expression of the proteinase inhibitor (0112A-3) and the protease (018-3); second, ozone stress may change the cell wall components by increasing the amount of glycine-rich proteins (OID2-2 and OIS14-3); and third, ozone stress may change the cell wall structure with tyrosine-mediated cross-linking, which may use oxyradical-scavenging effects to relieve cellular stress

Evaluation of the Ultraviolet-Curing Kinetics of Ultraviolet-Polymerized Oligomers Cured Using Poly (Ethylene Glycol) Dimethacrylate

Ultraviolet (UV)-curable oligomers are increasingly being used in various industries because they can be applied rapidly and have excellent physical properties. Ultraviolet polymerization is used for manufacturing such oligomers. Reactive diluents, which are employed during the secondary curing of UV-curable oligomers, can help elucidate the curing behaviors of these oligomers. In this study, poly (ethylene glycol) dimethacrylate (PEGDMA) was used as the reactive diluent for UV-curable oligomers. Photodifferential scanning calorimetry (photo-DSC) and shrinkage measurements revealed that the curing behavior of the polymers was dependent on the size and number of molecules of PEGDMA. The effect of the small-size PEGDMA on curing behavior was greater than that of the larger molecules. Further, in most cases, the use of a larger amount of PEGDMA resulted in lower reactivity

A Double-Strip Plasmonic Waveguide Coupled to an Electrically Driven Nanowire LED

We demonstrate the efficient integration of an electrically driven nanowire (NW) light source with a double-strip plasmonic waveguide. A top-down-fabricated GaAs NW light-emitting diode (LED) is placed between two straight gold strip waveguides with the gap distance decreasing to 30 nm at the end of the waveguide and operated by current injection through the p-contact electrode acting as a plasmonic waveguide. Measurements of polarization-resolved images and spectra show that the light emission from the NW LED was coupled to a plasmonic waveguide mode, propagated through the waveguide, and was focused onto a subwavelength-sized spot of surface plasmon polaritons at the tapered end of the waveguide. Numerical simulation agreed well with these experimental results, confirming that a symmetric plasmonic waveguide mode was excited on the top surface of the waveguide. Our demonstration of a plasmonic waveguide coupled to an electrically driven NW LED represents important progress toward further miniaturization and practical implementation of ultracompact photonic integrated circuits