Characterisation of microsatellite DNA markers for Grevillea paradoxa (F. Muell)
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MICROSATELLITE LETTERS
Characterisation of microsatellite DNA markers for Grevillea paradoxa (F. Muell) Melissa A. Millar • Margaret Byrne David J. Coates • J. Dale Roberts
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Received: 12 August 2013 / Accepted: 13 August 2013 / Published online: 28 August 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract A genomic library was constructed and 14 novel polymorphic di- and tri-nucleotide nuclear microsatellite markers were developed for Grevillea paradoxa, the Bottlebrush Grevillea, a common shrub in southwest Western Australia. Populations are patchily distributed in a highly fragmented landscape due to extensive vegetation clearing for agriculture and population genetic structure is being investigated to inform appropriate seed collection and restoration strategies. Moderate diversity was observed in two populations with the number of alleles per locus ranging from 1 to 9. Expected and observed heterozygosities averaged 0.434 and 0.344 respectively. All loci showed independent inheritance but there was evidence of possible null alleles at some loci in each population. Keywords Bottlebrush Grevillea Microsatellites M13 tagged primer Western Australia
Microsatellite markers were developed for Grevillea paradoxa, the Bottlebrush Grevillea. This common prickly shrub has a widespread distribution in species-rich southwest Western Australia, although populations are patchily M. A. Millar (&) M. Byrne D. J. Coates Science Division, Department of Parks and Wildlife, Locked Bag 104, Bentley Delivery Centre, Bentley, WA 6983, Australia e-mail: [email protected] M. A. Millar J. D. Roberts School of Animal Biology, Centre of Excellence in Natural Resource Management, The University of Western Australia, PO Box 5771, Albany, WA 6332, Australia M. Byrne D. J. Coates School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
distributed due to extensive vegetation clearing for agriculture. DNA was extracted from 40 mg of homogenised, freeze dried material from an individual originating near the centre of the species range, following the method of Doyle and Doyle (1987), with addition of PVP-40T 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 GS-FLX 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 15 ll per reaction containing 5 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 tailed forward primer, 0.15 ll of Taq DNA polymerase (Invitrogen) and 3.0 mM MgCl2. Amplifi
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