Isolation and characterisation of ten microsatellite markers for the tetraploid Stypandra glauca R.Br. (Hemerocallidacea
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TECHNICAL NOTE
Isolation and characterisation of ten microsatellite markers for the tetraploid Stypandra glauca R.Br. (Hemerocallidaceae) identified using next generation sequencing S.-L. Tapper • M. Byrne • M. G. Gardner
Received: 10 December 2012 / Accepted: 13 December 2012 / Published online: 21 December 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Ten polymorphic microsatellite loci were developed for the tetraploid plant species Stypandra glauca which is common on granite outcrops in Western Australia. Amplification and genotyping trials were conducted on 48 individuals from two sampling localities. All 10 loci revealed multi-banding patterns with up to 4 bands visible in individuals consistent with tetraploidy. The number of alleles per locus ranged from 6 to 32 (mean = 12.5). The proportion of observed heterozygotes at each locus ranged from 0.32 to 0.98 (mean = 0.73). These polymorphic microsatellite markers will facilitate further analysis of population genetic structure and connectivity in Stypandra glauca. Keywords Granite outcrop Refugia Western Australia Genetic diversity
Stypandra glauca is a small perennial herb species with a widespread distribution stretching from the South West
S.-L. Tapper (&) M. Byrne Science Division, Department of Environment and Conservation, Bentley Delivery Centre, Locked Bag 104, Kensington, WA 6983, Australia e-mail: [email protected]; [email protected] M. Byrne e-mail: [email protected] M. G. Gardner School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia e-mail: [email protected] M. G. Gardner Evolutionary Biology Unit, South Australian Museum, North Terrace, Adelaide, SA 5001, Australia
Botanical Province of Western Australia through to south east Queensland, Australia. Western Australian populations of S. glauca are considered to be autopolyploid due to the presence of univalents and irregular chromosome pairing during meiosis as well as substantial levels of pollen sterility (Russell 1988). In Western Australia, the species is commonly located in granite outcrop habitats. It has been hypothesized that these outcrops act as refugia for plants during periods of climatic aridity as they are water gaining sites and provide a range of microclimatic environments (Keppel et al. 2012). Analysis of genetic diversity and population structure in S. glauca is being incorporated into a multidisciplinary study of the role of granite outcrops as refugia. Here, microsatellite markers have been developed to determine patterns of genetic diversity and population structure in the nuclear genome of S. glauca, as microsatellite markers have proved valuable in assessing genetic structure in other polyploid species (Sampson and Byrne 2012; Clarke et al. 2012). Genomic DNA was extracted from freeze dried and ground leaf material. Extractions followed the Doyle and Doyle (1987) method with the addition of 1 % polyvinylpyrrolidone (PVP) to the extraction buffer. Four micrograms
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