Isolation of microsatellites using 454-sequencing for the grassland shrub Pimelea spinescens (Thymelaeaceae)
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TECHNICAL NOTE
Isolation of microsatellites using 454-sequencing for the grassland shrub Pimelea spinescens (Thymelaeaceae) R. Jordan • E. A. James • G. K. Brown A. L. Brown
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Received: 29 June 2012 / Accepted: 4 July 2012 / Published online: 21 July 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Ten microsatellite loci were developed for the grassland shrub Pimelea spinescens. Locus amplification varied between the two subspecies. For P. spinescens subsp. spinescens, nine loci amplified and were polymorphic. Number of alleles per locus ranged from two to eleven (average = 5.7) and expected heterozygosity ranged from 0.103 to 0.782. For P. spinescens subsp. pubiflora, seven loci amplified, of which only three were polymorphic. For polymorphic loci, number of alleles ranged from four to seven (average = 5.3) and expected heterozygosity ranged from 0.381 to 0.783. These loci will be used to investigate population genetic structure, assisting management of this endangered species. Keywords Dioecy Endangered Grassland Pimelea spinescens subspecies pubiflora Riceflower
Pimelea spinescens (Rye) is a mainly dioecious, long-lived shrub found in grassland and open shrubland of western and central Victoria, Australia (Walsh and Entwisle 1996). It comprises two subspecies, P. spinescens subsp. spinescens and P. spinescens subsp. pubiflora (hereafter ‘spinescens’ and ‘pubiflora’, respectively). Both subspecies are listed as Critically Endangered (EPBC Act 1999). ‘Spinescens’ is wide-spread but uncommon whereas ‘pubiflora’, believed extinct until rediscovered in 2005 R. Jordan E. A. James (&) G. K. Brown A. L. Brown Royal Botanic Gardens Melbourne, Birdwood Avenue, Private Bag 2000, South Yarra, Melbourne, VIC 3141, Australia e-mail: [email protected] E. A. James G. K. Brown School of Botany, The University of Melbourne, Melbourne, VIC 3010, Australia
(DSE 2006), is known only from two populations. With less than 1 % of pre-European settlement grassland believed to be remaining in south-eastern Australia (McDougall and Kirkpatrick 1994), both subspecies are under threat of further habitat loss. As part of a broader conservation plan for P. spinescens (Carter and Walsh 2006), microsatellites were developed to investigate the population genetics of this species. Genomic DNA was isolated from 120 mg of silica-dried leaf tissue of a single ‘spinescens’ plant using QIAGEN DNeasy Plant Minikit (QIAGEN Inc., Valencia, California). Next generation ‘454’ sequencing performed by the Australian Genome Research Facility (AGRF), with a 1/8 genome coverage, was used to isolate microsatellites. Raw sequences were searched for di- to hexa-nucleotide microsatellites, of minimum 8 repeats, using MSATCOMMANDER (Faircloth 2008) and unique loci using MICROFAMILY (Megle´cz 2007). Primers were designed with PRIMER3 (Rozen and Skaletsky 2000). Microsatellites were fluorescently labeled using a modified version of Shuelke (2000) described earlier (James et al. 2011; Blacket et al. 2012). Sixty-eight loci were
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