Characterisation of microsatellite DNA markers for Grevillea globosa C. A. Gardner
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MICROSATELLITE LETTERS
Characterisation of microsatellite DNA markers for Grevillea globosa C. A. Gardner Melissa A. Millar • Margaret Byrne David J. Coates • J. Dale Roberts
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Received: 3 March 2014 / Accepted: 27 March 2014 Ó Crown Copyright as represented by the Government of Western Australia, Department of Parks and Wildlife 2014
Abstract A genomic library was constructed and 14 novel polymorphic di- and tri-nucleotide nuclear microsatellite markers were developed for Grevillea globosa, an endemic shrub of southwest Western Australia. Populations are patchily distributed and population genetic structure is being investigated to inform appropriate seed collection and restoration strategies. Diversity in a selected population was high, with the number of alleles per locus ranging from 3 to 13 and expected and observed heterozygosities averaging 0.693 and 0.799 respectively. All loci showed independent inheritance and there was no evidence of possible null alleles. Keywords Australia
Microsatellites M13 tagged primer Western
Microsatellite markers were developed for Grevillea globosa, a shrub with a limited distribution in southwest M. A. Millar (&) M. Byrne D. J. Coates Science and Conservation Division, Department of Parks and Wildlife, Bentley Delivery Centre, Locked Bag 104, Bentley, WA 6983, Australia e-mail: [email protected] M. A. Millar School of Animal Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia M. Byrne D. J. Coates School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia J. D. Roberts School of Animal Biology, Centre for Evolutionary Biology and Centre of Excellence in Natural Resource Management, The University of Western Australia, PO Box 5771, Albany, WA 6332, Australia
Western Australia’s semiarid rainfall zone. Plants occur on sand and loam in Acacia shrubland, mallee woodland and shrubland, and Eucalypt woodland. Populations are patchily distributed and poorly known hence the taxon is of some conservation significance. DNA was extracted from homogenised, freeze dried material of an individual originating from the centre of the species range following the method of Doyle and Doyle (1987), with addition of PVP40T polyvinyl pyrrolidine to the extraction buffer and two chloroform extraction steps. Genomic DNA was 454 shotgun sequenced using 1\8 of a picotiter plate on a GSFLX Titanium machine (Roche Diagnostics Corporation, 454 Life Sciences, Branford, USA) by the Australian Genome Research Facility (Adelaide, Australia). Sequencing, microsatellite identification and primer design followed Gardner et al. (2011). Amplification and polymorphism were evaluated in eight individuals from four populations using an M13 tailed three primer PCR system. Loci were amplified in a total volume of 7.5 ll per reaction containing 2 ng template DNA, 50 mM KCl, 20 mM Tris HCl (pH 8.4), 0.2 mM each dNTP, 0.16 lM M13 tailed fluorescently labelled forward and reverse primer, 0.032 lM M13 t
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