Development of 10 Polymorphic Microsatellite Loci Isolated From The Mountain Beaver, \u3ci\u3eAplodontia rufa rufa\u3c/i\u3e (Rafinesque)

We developed 10 microsatellite markers for the mountain beaver, Aplodontia rufa rufa. In three populations of A. r. rufa, the number of alleles for these loci ranged from monomorphic to nine. Average observed heterozygosities in these populations ranged from 0.29 to 0.60. We also tested previously published markers from the endangered subspecies A. r. nigra in A. r. rufa populations

Genome-Enhanced Detection and Identification (GEDI) of plant pathogens

Plant diseases caused by fungi and Oomycetes represent worldwide threats to crops and forest ecosystems. Effective prevention and appropriate management of emerging diseases rely on rapid detection and identification of the causal pathogens. The increase in genomic resources makes it possible to generate novel genome-enhanced DNA detection assays that can exploit whole genomes to discover candidate genes for pathogen detection. A pipeline was developed to identify genome regions that discriminate taxa or groups of taxa and can be converted into PCR assays. The modular pipeline is comprised of four components: (1) selection and genome sequencing of phylogenetically related taxa, (2) identification of clusters of orthologous genes, (3) elimination of false positives by filtering, and (4) assay design. This pipeline was applied to some of the most important plant pathogens across three broad taxonomic groups: Phytophthoras (Stramenopiles, Oomycota), Dothideomycetes (Fungi, Ascomycota) and Pucciniales (Fungi, Basidiomycota). Comparison of 73 fungal and Oomycete genomes led the discovery of 5,939 gene clusters that were unique to the targeted taxa and an additional 535 that were common at higher taxonomic levels. Approximately 28% of the 299 tested were converted into qPCR assays that met our set of specificity criteria. This work demonstrates that a genome-wide approach can efficiently identify multiple taxon-specific genome regions that can be converted into highly specific PCR assays. The possibility to easily obtain multiple alternative regions to design highly specific qPCR assays should be of great help in tackling challenging cases for which higher taxon-resolution is needed

List of probes developed for each simplex, duplex and triplex assay, as identified in Fig 1.

<p>The first 3–4 letters of probe names designate the taxa being targeted (except for FS1, which designates the marker), while “RC” means reverse complement. In probe sequences, bases preceded by a “+” sign are LNA bases.</p

Example of a qPCR validation assay.

<p>Amplification curves obtained in validating the <i>L</i>. <i>albescens/L</i>. <i>postalba</i> assay tested on 61 samples (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.s001" target="_blank">S1 File</a>, “Duplex assay 1A” tab, for details on samples). Only <i>L</i>. <i>albescens</i> (red traces) and <i>L</i>. <i>postalba</i> (green traces) samples generated positive amplifications (results of three technical replicates are shown for each sample).</p

Amplification of undiluted and diluted (normalized) DNA samples.

<p>Comparison of amplification curves generated using COI lymantriine general primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.s001" target="_blank">S1 File</a>, tab 1) on multiple, undiluted samples <b>(A)</b>, and those obtained from the same samples following dilutions calculated to achieve the Ct of 22–23 <b>(B)</b>.</p

List of primers developed for DNA quantification and for each simplex, duplex and triplex assay, as identified in Fig 1.

<p>The first 3–4 letters of primer names designate the taxa being targeted (except for FS1, which designates the marker), while “F” and “R” refer to forward and reverse. In primer sequences, bold/underlined letters designate degenerate sites while italicized/underlined letters designate ARMS bases.</p

Specimens of target species and closely related ones used for assay development and validation.

<p>Specimens of target species and closely related ones used for assay development and validation.</p

Geographical distribution of FS1 genotypes, as determined using <i>Duplex assay 4</i>, for gypsy moths identified as <i>L</i>. <i>dispar dispar</i> using <i>Simplex assay 3</i>.

<p>Blue circles: homozygous for the FS1-N allele; red circles: homozygous for the FS1-A allele; blue/red circles: heterozygous for the N and A alleles. Black letters near each circle identify specimens identified as <i>L</i>. <i>dispar dispar</i> using <i>Simplex assay 3</i>; red letters designate <i>L</i>. <i>dispar asiatica</i> and <i>L</i>. <i>dispar japonica</i> positive controls (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.t004" target="_blank">Table 4</a> for details). Box: central Asia region rich in specimens that are homozygous for the FS1-A allele while displaying <i>L</i>. <i>dispar dispar</i> COI barcode. Background map is from <a href="https://commons.wikimedia.org/wiki/" target="_blank">https://commons.wikimedia.org/wiki/</a>.</p

A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (<i>Lymantria</i> spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America

<div><p>Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related <i>Lymantria</i> species (Lepidoptera: Erebidae: Lymantriinae) comprising two <i>L</i>. <i>dispar</i> subspecies (<i>L</i>. <i>dispar asiatica</i>, <i>L</i>. <i>dispar japonica</i>) and three closely related <i>Lymantria</i> species (<i>L</i>. <i>umbrosa</i>, <i>L</i>. <i>albescens</i>, <i>L</i>. <i>postalba</i>), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous <i>Lymantria</i> species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan^® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three <i>L</i>. <i>dispar</i> subspecies (including the European gypsy moth, <i>L</i>. <i>dispar dispar</i>), the three other <i>Lymantria</i> species comprising the AGM complex, plus five additional <i>Lymantria</i> species that pose a threat to forests in North America. The suite of assays is built as a “molecular key” (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5’ region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3’ region, as well as on the presence of an indel in the “FS1” nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into <i>L</i>. <i>dispar dispar</i>. These assays have the advantage of providing rapid and accurate identification of ten <i>Lymantria s</i>pecies and subspecies considered potential FIAS.</p></div