Soybean seeds overexpressing asparaginase exhibit reduced nitrogen concentration.

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K+ -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the ß-subunit of ß-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing ß-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11-18%, and an increase in free aspartic acid levels, by 25-60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9-13%. Their levels of extractible globulins were reduced by 11-30%. This was accompanied by an increase in oil concentration, by 5-8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation.

Resolving the Globisporangium ultimum (Pythium ultimum) species complex.

The Globisporangium ultimum (formerly Pythium ultimum) species complex was previously composed of two morphological varieties: var. ultimum and var. sporangiiferum. Prior attempts to resolve this morphology-based species complex using molecular techniques have been inconclusive or conflicting. The increased availability of sequenced genomes and isolates identified as G. ultimum var. ultimum and var. sporangiiferum has allowed us to examine these relationships at a higher resolution and with a broader scope than previously possible. Using comparative genomics, we identified highly variable gene regions and designed primers for four new protein-coding genes for phylogenetics. These were then used alongside three known markers to generate a nuclear multigene genealogy of the species complex. From a collection of 163 isolates belonging to the target taxa, a subset of 29 was chosen to be included in this study (verified with nuclear rDNA internal transcribed spacer 1 [ITS1] and mitochondrial cytochrome c oxidase subunit 1 [cox1] sequences). Seventeen isolates of var. ultimum were selected to be representative of variations in genotype, morphology, and geographic collection location. The 12 isolates of var. sporangiiferum included all available specimens identified either morphologically (in previous studies) or through sequence similarity with ITS1 and cox1. Based on the fulfillment of reciprocal monophyly and observed genealogical concordance under the genealogical concordance phylogenetic species recognition, we determined that the Globisporangium ultimum species complex is composed of four genetically distinct species: Globisporangium ultimum, Globisporangium sporangiiferum, Globisporangium solveigiae, and Globisporangium bothae.

Coloring genetically modified soybean grains with anthocyanins by suppression of the proanthocyanidin genes ANR1 and ANR2.

Detection and quantification of the levels of adventitious presence of genetically modified (GM) soybeans in non-GM grain shipments currently requires sophisticated tests that can have issues with their reproducibility. We show here that pigment biosynthesis in the soybean seed coat can be manipulated to provide a distinct color that would enable the simple visible detection of the GM soybean grain. We observed that a distinct red-brown grain color could be engineered by the simultaneous suppression of two proanthocyanidin (PA) genes, ANTHOCYANIDIN REDUCTASE1 (ANR1) and ANR2. Multiple reaction monitoring by liquid chromatography tandem mass spectrometry was used to quantify differentially accumulated seed coat metabolites, and revealed the redirection of metabolic flux into the anthocyanin pigment pathway and unexpectedly the flavonol-3-O-glucoside pathway. The upregulations of anthocyanin isogenes (DFR1 and GST26) and the anthocyanin/flavonol-3-O-glycosyltransferase (UGT78K2) were identified by quantitative RT-PCR to be endogenous feedback and feedforward responses to overaccumulation of upstream flavonoid intermediates resulting from ANR1 and ANR2 suppressions. These results suggested the transcription of flavonoid genes to be a key component of the mechanism responsible for the redirection of metabolite flux. This report identifies the suppression of PA genes to be a novel approach for engineering pigmentation in soybean grains.

Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium.

The genus Pythium (nom. cons.) sensu lato (s.l.) is composed of many important species of plant pathogens. Early molecular phylogenetic studies suggested paraphyly of Pythium, which led to a formal proposal by Uzuhashi and colleagues in 2010 to split the genus into Pythium sensu stricto (s.s.), Elongisporangium, Globisporangium, Ovatisporangium (= Phytopythium), and Pilasporangium using morphological characters and phylogenies of the mt cytochrome c oxidase subunit 2 (cox2) and D1-D2 domains of nuc 28S rDNA. Although the split was fairly justified by the delineating morphological characters, there were weaknesses in the molecular analyses, which created reluctance in the scientific community to adopt these new genera for the description of new species. In this study, this issue was addressed using phylogenomics. Whole genomes of 109 strains of Pythium and close relatives were sequenced, assembled, and annotated. These data were combined with 10 genomes sequenced in previous studies. Phylogenomic analyses were performed with 148 single-copy genes represented in at least 90% of the taxa in the data set. The results showed support for the division of Pythium s.l. The status of alternative generic names that have been used for species of Pythium in the past (e.g., Artotrogus, Cystosiphon, Eupythium, Nematosporangium, Rheosporangium, Sphaerosporangium) was investigated. Based on our molecular analyses and review of the Pythium generic concepts, we urge the scientific community to adopt the generic names Pythium, Elongisporangium, Globisporangium, and their concepts as proposed by Uzuhashi and colleagues in 2010 in their work going forward. In order to consolidate the taxonomy of these genera, some of the recently described Pythium spp. are transferred to Elongisporangium and Globisporangium.

Molecular phylogeny and taxonomy of Lagenidium-like oomycetes pathogenic to mammals.

Over the past twenty years, infections caused by previously unrecognised oomycete pathogens with morphological and molecular similarities to known Lagenidium species have been observed with increasing frequency, primarily in dogs but also in cats and humans. Three of these pathogens were formally described as Lagenidium giganteum forma caninum, Lagenidium deciduum, and Paralagenidium karlingii in advance of published phylogenetic verification. Due to the complex nature of Lagenidium taxonomy alongside recent reports of mammalian pathogenic species, these taxa needed to be verified with due consideration of the available data for Lagenidium and its allied genera. This study does so through morphologic characterisation of the mammalian pathogenic species, and phylogenetic analyses. The six-gene phylogeny generally supports the most recent comprehensive classification of Lagenidium with a well-supported Lagenidium clade that includes the mammalian pathogens L. giganteum f. caninum and L. deciduum, and well-supported clades for which the names Myzocytiopsis and Salilagenidium can be applied. The genus Paralagenidium is phylogenetically unrelated to any of the main clades within the class Peronosporomycetes. Close relationships between pathogens of mammals and those of insects or nematodes were revealed. Further characterisation of Lagenidium-like taxa is needed to establish the risk of mammalian infection by pathogens of insects and nematodes.

Multigene phylogenetic analyses to delimit new species in fungal plant pathogens.

Supporting the identification of unknown strains or specimens by sequencing a genetic marker commonly used for phylogenetics or DNA barcoding is now standard practice for mycologists and plant pathologists. Does one have a new species when a strain differs by a few base pairs when compared to reference sequences from taxonomically well-characterized species that do not differ morphologically from this new strain? If variation at the intra- and interspecific levels for the locus used for identification is already understood for all the closely related species, it is possible to make a reliable prediction of a new species status, but ultimately this question can only be properly addressed by determining the presence or absence of gene flow among a group of strains of the putative new species and strains of previously delimited species. The Phylogenetic Species Concept (PSC) and its assessment using multigene phylogeny and Genealogical Concordance Phylogenetic Species Recognition (GCPSR) are the basis for this chapter. The theoretical framework and a variety of tools to apply these concepts are explained, to assist in the assessment of whether a species is distinct or new when confronted with some sequence divergence from reference data.

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.