Genodive manual manual#

The 96 primer pairs were manufactured by IDT (Integrated DNA Technologies, Coralville, Iowa, USA) and screened for amplification and variability in three D. A total of 96 loci were selected for screening based on penalty score (lowest score indicates “best” primer pair at the locus), size of DNA fragment (100–400 bp), and motif repeat size.

The program QDD was used to search for tandem repeats and to design microsatellite primers ( Meglécz et al., 2009), resulting in the isolation of 11,480 possible microsatellite loci. DNA was sequenced for primer development using a Roche GS FLX machine for a 1/8 coverage (454 Life Sciences, Branford, Connecticut, USA). The final quality of the library was assessed using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, California, USA) and quantitative PCR (qPCR) using KAPA reagents (KAPA Biosystems, Woburn, Massachusetts, USA). Small fragments were removed using Agencourt AMPure Beads (Beckman Coulter, Lane Cove, New South Wales, Australia). Fragments underwent end repair and subsequent barcoded adapter ligation with the 454 Adapter-A and Adapter-B (Lib-L). The DNA was nebulized and cleaned with a QIAGEN MiniElute purification kit.

Genodive manual manual#

The genomic DNA library was prepared according to Roche's Rapid Library Preparation Method Manual and quantified by PicoGreen fluorimetry (Quant-iT PicoGreen dsDNA Assay Kit Invitrogen, Carlsbad, California, USA). basaltica individual using a DNeasy Plant Maxi Kit (QIAGEN, Hilden, Germany). Genomic DNA was extracted and isolated from 300 mg of silica-dried leaf tissue from one D. The other three populations used in this study were identified as D. Of the >100 cultivated plants, 18 were sampled for this survey. METHODS AND RESULTSĭiuris basaltica was sampled from known collections that were cultivated in a greenhouse at Royal Botanic Gardens Melbourne. In addition to assessment of current species delimitation, the markers will help with future reintroduction efforts by enabling any changes in genetic structure, such as possible genetic erosion, to be monitored over time. The markers were developed using 454 pyrosequencing because next-generation sequencing reduces total cost, provides rapid development in nonmodel organisms, is not limited to particular repeat motifs, and locates thousands of possible microsatellite loci, which increases the success of finding polymorphic loci ( Schoebel et al., 2013). Genetic markers are needed to investigate the legitimacy of Diuris species, understand how populations are connected, and aid in the conservation of D. Therefore, the use of current genetic techniques and morphological measurements should both be used to delimit species within Orchidaceae genera. This suggests that the morphology used to delimit Orchidaceae species may not necessarily reflect the genetic composition of populations and may mislead conservation efforts because the inclusion of poorly substantiated taxa could be a hindrance to conservation plans. (2009) also conclude that gene flow between geographically close populations is more likely than between morphologically similar ones at greater distances. It has been established in other Orchidaceae genera that genetics do not always follow current species delimitations ( Devey et al., 2008, 2009), and the Diuris complex may be no different. basaltica occur in the region and have subtle morphological differences ( D. At least three species closely related to D. Jones (Orchidaceae) occurs within this region and is only known to occur in three locations ( Backhouse and Lester, 2010) however, the erratic flowering patterns make this difficult to confirm even though it was once “relatively frequent” in areas just east of Melbourne ( Jones, 2006). Remnants in the VVP house the remaining endemic and naturally occurring species, many of which are in decline. The Natural Temperate Grassland of the Victorian Volcanic Plain (VVP) of Victoria, Australia, has been under constant anthropogenic pressure over the past century ( McDougall and Kirkpatrick, 1994).