The discovery of microsatellite markers for Hardenbergia violacea (Fabaceae), using next-generation sequencing
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TECHNICAL NOTE
The discovery of microsatellite markers for Hardenbergia violacea (Fabaceae), using next-generation sequencing M. Fatemi • A. Haddadchi • C. L. Gross
Received: 22 May 2012 / Accepted: 25 June 2012 / Published online: 5 July 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Hardenbergia violacea is a climbing to prostrate shrub endemic to woodlands in the south east of Australia. We used next generation sequencing to develop a species-specific primer set. We obtained 106,529 reads in FASTA format with an average read length of 548 bp. DNA was successfully amplified for 20 markers and eight markers were characterized. The degree of polymorphism of these loci was tested on 95 samples from five populations. The total number of alleles ranged from two to 11 and observed and expected heterozygosities ranged from 0.0 to 0.807 and 0.0 to 0.933, respectively. Inbreeding coefficients were highly variable among populations, suggesting a mixture of selfing and outcrossing in nature. The characterized loci will be useful for studies of population structure, gene flow, mating systems, and the conservation and selection of provenances for restoration projects in degraded habitats. Keywords
454 GS-FLX Mating system SSRs
Hardenbergia violacea (Schneev.) Stearn is a sprawling shrub endemic to south eastern Australia. It is found in dry open sclerophyll forests and coastal heaths. It is a common species on the mainland but is considered a threatened species in Tasmania (Larcombe et al. 2011; Threatened Species Section 2009).
M. Fatemi (&) A. Haddadchi C. L. Gross Ecosystem Management, University of New England, Armidale, NSW 2351, Australia e-mail: [email protected]
Microsatellites have become important genetic markers for the study of mating systems, conservation genetics, and evolutionary biology (Conte et al. 2008), because they are highly polymorphic and can be scored relatively easily (Zane et al. 2002). However, the major drawback of traditional approaches to develop microsatellites is that the de novo generation of markers is highly time-consuming and/or expensive due to the requirement to enrich genomic DNA for repeated motifs, cloning and sequencing of target fragments that contain microsatellite motifs. The identification of microsatellite markers using next generation sequencing is a fast and cost-effective approach which is becoming more popular with the fall of sequencing costs (Zalapa et al. 2012; Fatemi et al. 2012). In this paper, we describe the development of species-specific microsatellite loci for H. violacea using 454 sequencing. Our aim was to develop and test microsatellite markers by combining the 454 sequencing with the screening of selected markers using the QIAxcel gel electrophoresis system before labelling with cost-effective M13 fluorophore-conjugated primers (Schuelke 2000). Leaf samples were collected from five populations in eastern Australia (Putty Road, PR, n = 23; Wallaby Scrub Road-1, WS-1, n = 12; and Wallaby Scrub Road-2, WS-2 n = 17), Copeton Dam (CD, n = 15
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