Isolation and characterization of 11 polymorphic microsatellite markers in the highly invasive Western conifer seed bug,
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MICROSATELLITE LETTERS
Isolation and characterization of 11 polymorphic microsatellite markers in the highly invasive Western conifer seed bug, Leptoglossus occidentalis (Heteroptera, Coreidae) V. Lesieur • B. Courtial • A. Roques M. A. Auger-Rozenberg
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Received: 13 January 2014 / Accepted: 31 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Eleven polymorphic microsatellite markers were developed from enriched DNA libraries for the invasive Western conifer seed bug, Leptoglossus occidentalis. The number of alleles ranged from 2 to 11 and observed heterozygosities from 0.038 to 0.933. Additional results of cross-species amplifications are reported for two congeneric species. This set of microsatellite markers, the first one available for L. occidentalis, enables further investigations of population structure of this species which represents a serious threat for European conifer regeneration. Keywords
Genetic diversity Invasion Microsatellites
The Western conifer seed bug, Leptoglossus occidentalis Heidemann (Heteroptera, Coreidae), was accidentally introduced in Europe and first reported in Italy in 1999 (Fent and Kment 2011). Then, the bug colonized most of Europe within just a decade (Fent and Kment 2011). Adults and nymphs feed on cones of a wide range of conifer species. Consequently, this introduction represents a risk not only for commercial seed crops but also for conifer ecosystems, impacting natural regeneration (Lesieur et al. 2014). The impact of L. occidentalis can also be enhanced through a newly established association between the alien insect and a native fungal pathogen, Diplodia pinea (Luchi et al. 2012). In order to set up an appropriate management program in Europe, microsatellites are needed to reveal the origin of newly established populations. We report here the isolation and characterization of 11 microsatellite loci useful for estimating genetic diversity in L. occidentalis. V. Lesieur (&) B. Courtial A. Roques M. A. Auger-Rozenberg UR633, Zoologie Forestie`re, INRA, 45075 Orle´ans, France e-mail: [email protected]
Total genomic DNA was isolated from one pooled sample of individuals collected from four localities situated in France (Lavercantie`re, Southwestern France and Serre-Ponc¸on, French Alps) and in the native range (two different sites in British Columbia, Canada). The extraction was then sent to GenoScreen, France (www.genosc reen.com). A total of 1 lg was used for the development of microsatellite libraries through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries, as described in Malausa et al. (2011). Among 907 sequences comprising a microsatellites motif, 298 primer sets were designed and a sub-group of 48 primers pairs was tested for amplification. Primer sets were discarded if they failed to amplify or led to multiple fragments. Then, 12 microsatellites loci were selected from validated ones for polymorphism study. PCR amplifications were performed in a volume of 25 ll containing 20 ng of template DNA, 1 U of Dre
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