Characterisation of 18 microsatellite markers for the thrips Cycadothrips chadwicki (Thysanoptera: Aeolothripidae), an o
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Characterisation of 18 microsatellite markers for the thrips Cycadothrips chadwicki (Thysanoptera: Aeolothripidae), an obligate mutualist pollinator of 11 Macrozamia cycad species Dean R. Brookes • James P. Hereward
Received: 10 June 2013 / Accepted: 21 June 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Several cycad species in the genus Macrozamia are threatened by habitat loss, fragmentation and pollinator loss including at least 11 that rely on the thrips Cycadothrips chadwicki for pollination and therefore their continued survival. We developed 18 highly polymorphic microsatellite markers (mean alleles per locus = 11.1) by 454 shotgun sequencing and present primer options for an additional 14,219 loci. Keywords Thrips Cycads Thysanoptera Macrozamia Microsatellite 454 pyrosequencing
Cycadothrips chadwicki (Mound 1991) is currently regarded as a single species of thrips that shares an obligate pollination mutualism with at least 11 Macrozamia cycad species. Many of these cycads are under threat of habitat loss, fragmentation and pollinator loss (Terry et al. 2008) with two classed as near threatened and two as vulnerable (IUCN 2012). Furthermore, sequence data indicates that there are likely cryptic species within C. chadwicki (D. R. Brookes, unpublished data). Understanding the species status, host associations, and extent of gene flow in
C. chadwicki is therefore critical for the conservation of both the pollinator and the cycad hosts that rely on them for reproduction. Here we report the development of 18 microsatellite markers for C. chadwicki, 10 of which amplify across multiple putative cryptic species. 100 individuals were taken from a single female cone of Macrozamia lucida collected in Brisbane Forest Park (QLD, Australia) and DNA was extracted from this pool (to provide sufficient quantity) using DNeasy columns (Qiagen). The DNA pool was shotgun sequenced on 454 plate by Macrogen (Korea), resulting in 439,442 reads (mean length = approx. 490 bp.) Reads under 80 bp were removed by the QDD algorithm (Megle´cz et al. 2010) leaving 415,239 reads. Microsatellites were then selected and screened for possible transposable copies with the QDD program; 13.42 % of these reads contained a microsatellite over 5 repeats. The QDD algorithm successfully designed primers for 14,303 loci using Primer3 (Rozen and Skaletsky 2000). Dinucleotides were the most common repeat (Fig. 1) but there were 1,272 unique tetranucleotides. For easier scoring we thus restricted our choice of primers to tetranucleotide and pentanucleotide microsatellites.
Electronic supplementary material The online version of this article (doi:10.1007/s12686-013-9993-6) contains supplementary material, which is available to authorized users. D. R. Brookes (&) J. P. Hereward School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia e-mail: [email protected]
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Conservation Genet Resour Fig. 1 Distribution of repeat motifs in shotgun sequence of C.
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