Isolation and characterisation of polymorphic microsatellite markers in the western ringtail possum, Pseudocheirus occidentalis

Abstract

We report on the development of 14 novel polymorphic microsatellite markers from the western ringtail possum (Pseudocheirus occidentalis), a ''threatened'' (Vulnerable) arboreal marsupial, endemic to the biodiversity hotspot region of south-west of Western Australia. The markers were developed to obtain population genetic data to quantify any changes which may be associated with increasing geographical fragmentation. We describe the diversity among 66 unrelated (adult) P. occidentalis individuals. Overall, the markers produced between three to eight alleles per locus and observed and expected heterozygosities of 59% and 69%, respectively. Due to predation by introduced foxes, urban encroachment changes in fire regime and the resultant levels of fragmentation in its range, these microsatellite markers are an important tool for evaluating population genetic information and for prioritising populations for conservation management.The western ringtail possum (Pseudocheirus occidentalis) is a medium sized nocturnal marsupial weighing up to 1.3 kg. It is endemic to south-western Australia (Van Dyck and Strahan 2008). The species is under increasing threat from introduced (cats and foxes) and native (pythons and chuditch) predators, changes in fire management practices and habitat fragmentation (Van Dyck and Strahan 2008). It has suffered localised decline over much of its range and localised extinction from several inland locations. As a result, understanding the spatial distribution and identifying populations of high conservation value (from a genetic point of view) will be crucial for development of effective management measures. It has previously proven difficult to obtain polymorphic markers for the western ringtail possum and none of the primers for the brushtail possum (Spencer and Bryant 2001, Taylor and Cooper 1998Hansen et al. 2005, Mitrovski et al. 2005 were found to amplify in the ringtail possum. As such it was necessary to find a species-specific alternative. Here we report on the development of polymorphic microsatellite loci which provide sufficient resolution for detecting fine-scale population genetic structure, gene flow and measures of population genetic diversity.Ninety-eight plasmid clones were sequenced (Genebank accession numbers GQ222585-GQ222683) and 30 microsatellite-containing regions were selected based on a minimum of 200 base pairs (bp) of high sequence-quality flanking either side of the repetitive element to design primers. Primer Express (Version 2.0; Applied Biosystems) was used to design a set of primers for each of the 30 sequences, using the following parameters: (i) Tm between 57°C and 63°C, (optimal 59°C); (ii) GC content of 45-55%; a 3 0 GC clamp of 3 residues; (iii) primer length 20-26 bp (optimum 24); and (iv) the PCR amplicon size of 80-350 bp. Primer pairs for these 30 sequences were ordered with an M13 tag (Schuelke 2000) and optimised to work in a 30 ll volume temperature-gradient PCR (55-65°C) containing 19 reaction buffer, 2.5 mM MgCl 2 , 0.1 mM bovine serum

Inhibition of α-Amylase and Glucoamylase by Tannins Extracted from Cocoa, Pomegranates, Cranberries, and Grapes

“…Additionally, the sharp doublet signal near 7.5 ppm (and a small broad signal at the same position for the grape specimen) was not present in spectra for cranberry and cocoa specimens and is consistent with hydrolyzable ellagic and/or gallic acid. 33…”

Limited ecological connectivity of an arboreal marsupial across a forest/plantation landscape despite apparent resilience to fragmentation

“…2009), three originated from the greater glider, Petauroides volans (Taylor et al. 2002) and three were sourced from the closely related western ringtail possum, Pseudocheirus occidentalis (Wilson et al. 2009).…”

“…HH, Hell's Hole; HS, Honeysuckle; SG, Snowgum; WD, Wild Dog. 16 microsatellite loci, of which 10 had been previously isolated from the species (Lancaster et al 2009), three originated from the greater glider, Petauroides volans (Taylor et al 2002) and three were sourced from the closely related western ringtail possum, Pseudocheirus occidentalis (Wilson et al 2009). Loci were labelled on the forward primer with a fluorescent dye tag (Applied Biosystems) and amplified, visualized and scored according to Lancaster et al (2009).…”

An Endangered Arboreal Specialist, the Western Ringtail Possum (Pseudocheirus occidentalis), Shows a Greater Genetic Divergence across a Narrow Artificial Waterway than a Major Road

“…Concentration and quality of each DNA sample were then determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Massachusetts, USA). We used 12 species-specific microsatellite markers (A1, A106, A119, A122, A127, A2, A6, B104, C111, D104, D113, and D114) following PCR conditions described by Wilson et al [ 21 ]. Genotypes at each locus were determined using an ABI 3700 Genetic Analyzer with a GeneScan-500 LIZ dye size standard (Applied Biosystems Inc., California, USA).…”