Development of microsatellite markers for Virola officinalis Warb. (Myristicaceae), an endemic tree of the Brazilian Atl
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MICROSATELLITE LETTERS
Development of microsatellite markers for Virola officinalis Warb. (Myristicaceae), an endemic tree of the Brazilian Atlantic rain forest by next generation sequencing technology Flora Bittencourt • Roberto Tarazi Fernanda A. Gaiotto
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Received: 5 July 2013 / Accepted: 19 July 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract We report the development and characterization of microsatellite markers for Virola officinalis, an endemic Brazilian Atlantic rain forest tree and important food resource for local wildlife. Seventy-two individuals were collected to characterize the 21 primers developed by next generation sequencing. Of the 21 primers studied, 15 were polymorphic. On average, the expected heterozygosity per population ranged from 0.64 to 0.73. The combined probability of exclusion was higher than 0.999, indicating that the microsatellite markers developed are suitable for genetic structure investigations and paternity testing. Our results will contribute to the design of efficient conservation strategies not only for this species, but for the Atlantic rain forest as a whole. Keywords NGS Primer Conservation genetics Endangered species
Virola officinalis Warb. (Myristicaceae) is an endemic woody tree from the Atlantic rain forest (Rodrigues 1980). Its fruits are sought by large birds and other endemic and endangered animals which use it as a food resource and act as seed dispersers. Arboreal species from Atlantic rain forest are now threatened with extinction because of habitat reduction, selective logging, and defaunation (Martini 2007). Diversity studies of species populations are useful in
F. Bittencourt R. Tarazi F. A. Gaiotto (&) Laboratory of Molecular Markers, Center of Biotechnology and Genetics (CBG), State University of Santa Cruz (UESC), Rodovia Ilhe´us-Itabuna Km 16 s/n, Salobrinho, Ilhe´us, Bahia 45662-900, Brazil e-mail: [email protected]
assessing the effects of human activities on ecosystems and planning conservation strategies. Taking this into consideration, we developed 21 microsatellite markers for V. officinalis using an enrichment genomic library with the probes (AC)n and (AG)n and ultrafast, large scale nextgeneration sequencing (454 pyrosequencing technology, Ecogenics GmbH, Schlieren, Switzerland). This sequencing tool is useful for elucidating the molecular genetic structure and evolutionary potential of target species (Ekblom and Galindo 2011). The enriched genomic library was constructed with DNA from young, fresh leaves from a single V. officinalis individual located in the Private Reserve, Nova Angelica (15°140 5300 S, 39°40 4500 W). A total of 172 sequences were generated between 82 and 218 bp. Of these, 15.11 % had microsatellites. Twenty-four pairs of primers were designed, three of these did not produce amplicons and were excluded from the study. The remaining 21 pairs of primers exhibited patterns of amplification relevant to the expected fragment sizes, but only 15 were polymorphic. The annealing temperature ranged from 52 t
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