Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data.

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217,000 reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. Eagleview software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability.

[Transmission of agents of the porcine respiratory disease complex (PRDC) between swine herds: a review. Part 2--Pathogen transmission via semen, air and living/nonliving vectors]. / Die Ubertragung von Erregern des porcine respiratory disease complex (PRDC) zwischen Schweineherden--eine Literaturübersicht. 2. Mitteilung--Ubertragung durch Sperma, Luft und belebte/unbelebte Vektoren.

The transmission of PRDC-pathogens (PRRSV, influenza virus A, PCV2, M. hyopneumoniae, A. pleuropneumoniae) between swine herds, which was summarized in the first part of the review, mainly occurs via pig movement. The risk of pathogen transmission by insemination with contaminated semen plays only a relevant role in the infection with PRRSV and PCV2. A risk of the aerogen transmission of pathogens between herds within a distance of 2 to 3 km is described for M. hyopneumoniae and PRRSV. Evidence for the other pathogens is not investigated. The PRDC-pathogens are frequently detected in wild boar populations. Therefore, the transmission between wild boars and domestic pigs seems possible by close contacts. PRRSV and M. hyopneumoniae can be transmitted by contaminated clothes and boots, but the use of sanitation protocols appears to limit their spread. Live vectors like rodents or birds seemed to have no special importance for the transmission of PRDC-pathogens.

[Transmission of agents of the porcine respiratory disease complex (PRDC) between swine herds: a review. Part 1--diagnosis, transmission by animal contact]. / Die Ubertragung von Erregern des porcine respiratory disease complex (PRDC) zwischen Schweineherden--eine Literaturübersicht. 1. Mitteilung--Diagnostik, Ubertragung durch Tierkontakte.

Knowledge on the different ways of transmitting PRDC pathogens (PRRSV, influenza virus A, PCV 2, M. hyopneumoniae, A. pleuropneumoniae) between swine herds is of special interest for the development of biosecurity measures or the retrospective risk analysis in the framework of activities of the consulting veterinarian. In this literature review the current knowledge of the transmission of PRDC-pathogens is summarized. Since the assessment of investigations into pathogen detection in detail is influenced considerably by the chosen test for the diagnosis, the standard methods of routine diagnostic procedures are described. In this context the limits of the interpretation of the diagnostic findings are especially described in detail. Finally, the transmission caused by pig movement is summarized in this first part of the review.

Thirty polymorphic nuclear microsatellite loci from black walnut.

Black walnut (Juglans nigra L) is a large tree, native to the eastern United States, that is prized for its high-quality timber and edible nut. Thirty (GA/CT)n nuclear microsatellite markers were identified from black walnut for use in population genetic studies, genome mapping, DNA genotyping of important clones, studies of gene flow, and tree breeding. The markers were polymorphic based on a diversity panel of 10 black walnut individuals from eight Midwestern U.S. states.

A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype.

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

Two Arabidopsis mutants that overproduce ethylene are affected in the posttranscriptional regulation of 1-aminocyclopropane-1-carboxylic acid synthase.

The Arabidopsis mutants eto1 (ethylene overproducer) and eto3 produce elevated levels of ethylene as etiolated seedlings. Ethylene production in these seedlings peaks at 60 to 96 h, and then declines back to almost wild-type levels. Ethylene overproduction in eto1 and eto3 is limited mainly to etiolated seedlings; light-grown seedlings and various adult tissues produce close to wild-type amounts of ethylene. Several compounds that induce ethylene biosynthesis in wild-type, etiolated seedlings through distinct 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) isoforms were found to act synergistically with eto1 and eto3, as did the ethylene-insensitive mutation etr1 (ethylene resistant), which blocks feedback inhibition of biosynthesis. ACS activity, the rate-limiting step of ethylene biosynthesis, was highly elevated in both eto1 and eto3 mutant seedlings, even though RNA gel-blot analysis demonstrated that the steady-state level of ACS mRNA was not increased, including that of a novel Arabidopsis ACS gene that was identified. Measurements of the conversion of ACC to ethylene by intact seedlings indicated that the mutations did not affect conjugation of ACC or the activity of ACC oxidase, the final step of ethylene biosynthesis. Taken together, these data suggest that the eto1 and eto3 mutations elevate ethylene biosynthesis by affecting the posttranscriptional regulation of ACS.

The molecular basis of ethylene signalling in Arabidopsis.

The simple gas ethylene profoundly influences plants at nearly every stage of growth and development. In the past ten years, the use of a genetic approach, based on the triple response phenotype, has been a powerful tool for investigating the molecular events that underlie these effects. Several fundamental elements of the pathway have been described: a receptor with homology to bacterial two-component histidine kinases (ETR1), elements of a MAP kinase cascade (CTR1) and a putative transcription factor (EIN3). Taken together, these elements can be assembled into a simple, linear model for ethylene signalling that accounts for most of the well-characterized ethylene mediated responses.

Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively.

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

Isolation and characterization of Arabidopsis mutants defective in the induction of ethylene biosynthesis by cytokinin.

Cytokinins elevate ethylene biosynthesis in etiolated Arabidopsis seedlings via a post-transcriptional modification of one isoform of the key biosynthetic enzyme ACC synthase. In order to begin to dissect the signaling events leading from cytokinin perception to this modification, we have isolated a series of mutants that lack the ethylene-mediated triple response in the presence of cytokinin due to their failure to increase ethylene biosynthesis. Analysis of genetic complementation and mapping revealed that these Cin mutants (cytokinin-insensitive) represent four distinct complementation groups, one of which, cin4, is allelic to the constitutive photomorphogenic mutant fus9/cop10. The Cin mutants have subtle effects on the morphology of adult plants. We further characterized the Cin mutants by analyzing ethylene biosynthesis in response to various other inducers and in adult tissues, as well as by assaying additional cytokinin responses. The cin3 mutant did not disrupt ethylene biosynthesis under any other conditions, nor did it disrupt any other cytokinin responses. Only cin2 disrupted ethylene biosynthesis in multiple circumstances. cin1 and cin2 made less anthocyanin in response to cytokinin. cin1 also displayed reduced shoot initiation in tissue culture in response to cytokinin, suggesting that it affects a cytokinin signaling element.