A Multiplex TaqMan qPCR Assay for Detection and Quantification of Clade 1 and Clade 2 Isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli.

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudoperonospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens' mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.

Assembly, annotation, and comparison of Macrophomina phaseolina isolates from strawberry and other hosts.

BACKGROUND: Macrophomina phaseolina is a fungal plant pathogen with a broad host range, but one genotype was shown to exhibit host preference/specificity on strawberry. This pathogen lacked a high-quality genome assembly and annotation, and little was known about genomic differences among isolates from different hosts. RESULTS: We used PacBio sequencing and Hi-C scaffolding to provide nearly complete genome assemblies for M. phaseolina isolates representing the strawberry-specific genotype and another genotype recovered from alfalfa. The strawberry isolate had 59 contigs/scaffolds with an N50 of 4.3 Mb. The isolate from alfalfa had an N50 of 5.0 Mb and 14 nuclear contigs with half including telomeres. Both genomes were annotated with MAKER using transcript evidence generated in this study with over 13,000 protein-coding genes predicted. Unique groups of genes for each isolate were identified when compared to closely related fungal species. Structural comparisons between the isolates reveal large-scale rearrangements including chromosomal inversions and translocations. To include isolates representing a range of pathogen genotypes, an additional 30 isolates were sequenced with Illumina, assembled, and compared to the strawberry genotype assembly. Within the limits of comparing Illumina and PacBio assemblies, no conserved structural rearrangements were identified among the isolates from the strawberry genotype compared to those from other hosts, but some candidate genes were identified that were largely present in isolates of the strawberry genotype and absent in other genotypes. CONCLUSIONS: High-quality reference genomes of M. phaseolina have allowed for the identification of structural changes associated with a genotype that has a host preference toward strawberry and will enable future comparative genomics studies. Having more complete assemblies allows for structural rearrangements to be more fully assessed and ensures a greater representation of all the genes. Work with Illumina data from additional isolates suggests that some genes are predominately present in isolates of the strawberry genotype, but additional work is needed to confirm the role of these genes in pathogenesis. Additional work is also needed to complete the scaffolding of smaller contigs identified in the strawberry genotype assembly and to determine if unique genes in the strawberry genotype play a role in pathogenicity.

Variable gene transcription underlies phenotypic convergence of hypoxia tolerance in sculpins.

BACKGROUND: The degree by which mechanisms underlying phenotypic convergence are similar among taxa depends on the number of evolutionary paths available for selection to act upon. Likelihood of convergence will be influenced by an interplay of factors such as genetic architecture, phylogenetic history and population demography. To determine if there is convergence or divergence in mechanisms underlying phenotypic similarity, we assessed whether gene transcription patterns differed among species with similar levels of hypoxia tolerance. RESULTS: Three species of marine fish from the superfamily Cottoidea (smoothhead sculpin [Artedius lateralis], sailfin sculpin [Nautichthys oculofasciatus] and Pacific staghorn sculpin [Leptocottus armatus]), all of which have previously been shown to share the same level of hypoxia tolerance, were exposed to short-(8 h) and longer-term (72 h) hypoxia and mRNA transcripts were assessed using a custom microarray. We examined hypoxia-induced transcription patterns in metabolic and protein production pathways and found that a high proportion of genes associated with these biological processes showed significant differences among the species. Specifically, the data suggest that the smoothhead sculpin, unlike the sailfin sculpin and the Pacific staghorn sculpin, relied on amino acid degradation rather than glycolysis or fatty acid oxidation to generate ATP during hypoxia exposure. There was also variation across the species in the transcription of genes involved in protein production (e.g. mRNA processing and protein translation), such that it increased in the smoothhead sculpin, decreased in the sailfin sculpin and was variable in the Pacific staghorn sculpin. CONCLUSIONS: Changes in metabolic and protein production pathways are part of the key responses of fishes to exposures to environmental hypoxia. Yet, species with similar overall hypoxia tolerance exhibited different transcriptional responses in these pathways, indicating flexibility and complexity of interactions in the evolution of the mechanisms underlying the hypoxia tolerance phenotype. The variation in the hypoxia-induced transcription of genes across species with similar hypoxia tolerance suggests that similar whole-animal phenotypes can emerge from divergent evolutionary paths that may affect metabolically important functions.

Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and Soil.

Macrophomina phaseolina is a broad-host-range fungus that shows some degree of host preference on strawberry, and causes symptoms that include crown rot and root rot. Recently, this pathogen has affected strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan polymerase chain reaction assay which is able to quantify as little as 2 to 3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.

Divergent transcriptional patterns are related to differences in hypoxia tolerance between the intertidal and the subtidal sculpins.

Transcriptionally mediated phenotypic plasticity as a mechanism of modifying traits in response to an environmental challenge remains an important area of study. We compared the transcriptional responses to low oxygen (hypoxia) of the hypoxia-tolerant intertidal fish, the tidepool sculpin (Oligocottus maculosus) with the closely related hypoxia-intolerant subtidal fish, the silverspotted sculpin (Blepsias cirrhosus) to determine whether these species use different mechanisms to cope with hypoxia. Individuals from each species were exposed to environmental O(2) tensions chosen to yield a similar level of tissue hypoxia, and gene transcription was assessed in the liver over time. There was an effect of time in hypoxia, where the greatest transcriptional change in the silverspotted sculpin occurred between 3 and 24 h in contrast to the tidepool sculpin where the largest transcriptional change occurred between 24 and 72 h of hypoxia. A number of genes showed similar hypoxia-induced transcription patterns in both species (e.g. genes associated with glycolysis and apoptosis) suggesting they are involved in a conserved hypoxia response. A large set of genes showed divergent transcriptional patterns in the two species, including fatty acid oxidation and oxidative phosphorylation, suggesting that these biological processes may contribute to explaining variation in hypoxia tolerance in these species. When both species were exposed to a single environmental O(2) tension, large transcriptional responses were seen in the hypoxia-intolerant silverspotted sculpin while almost no response was observed in the hypoxia-tolerant tidepool sculpin. Overall, divergent transcription patterns in response to both magnitude and duration of hypoxia provide insights into the processes that may determine an animal's capacity to tolerate frequent bouts of hypoxia in the wild.

Molecular support for marine sculpin (Cottidae; Oligocottinae) diversification during the transition from the subtidal to intertidal habitat in the Northeastern Pacific Ocean.

Sculpins in the genera Ruscarius, Artedius, Clinocottus, and Oligocottus are common intertidal and subtidal benthic fishes of the Northeastern Pacific Ocean. While there has been a long history of attempts to reconstruct the evolutionary relationships within this clade, studies have largely resulted in conflicting conclusions. Current ideas regarding the limits of species and genera in this subfamily (Oligocottinae) and their branching order are based primarily on morphology [Bolin, R.L., 1944. A Review of the Marine Cottid Fishes of California. Natural History Museum of Stanford [corrected] University, Stanford [corrected] University, California; Bolin, R.L., 1947. The Evolution of the Marine Cottidae of California with a Discussion of the Genus as a Systematic Category. Stanford [corrected] University, California]. The primary objectives of this study are: (a) to determine if the phylogenetic relationships inferred from DNA characters are concordant with those inferred from morphological characters and (b) to determine if a habitat shift from the subtidal to the intertidal environment resulted in the diversification of the group. Cytochrome b and Nicotinamide Adenine Dinucleotide Dehydrogenase subunit one mitochondrial gene fragments and one nuclear intron (S7 ribosomal protein) were sequenced in order to infer the phylogenetic relationships within this subfamily. Maximum likelihood and Bayesian algorithms were employed to reconstruct phylogenetic trees. We found that several genera in this clade are not monophyletic and that there is a clear phylogenetic signal indicating that a habitat shift from the subtidal to the intertidal habitat has resulted in the diversification of the Oligocottinae.

Lack of mitochondrial genetic structure in hamlets (Hypoplectrus spp.): recent speciation or ongoing hybridization?

Species in the genus Hypoplectrus (hamlet fish) have been recognized primarily on the basis of colour morphology, which varies substantially. Limited differentiation in other morphological characters, however, has led to ongoing debate about their taxonomy. Our analysis of mtDNA sequences demonstrates neither reciprocal monophyly nor significant genetic differentiation among hamlet colour morphs. These data are potentially consistent with a model of recent speciation due to sexual selection on colour morphology. The presence within hamlets of two divergent mtDNA clades, however, suggests a longer history during which hybridization and gene flow have prevented the differentiation of hamlet colour morphs, at least in neutral genetic markers.