Development of microsatellite primers for Melanoxylon brauna (Fabaceae): an endangered and endemic tree from the Brazili
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MICROSATELLITE LETTERS
Development of microsatellite primers for Melanoxylon brauna (Fabaceae): an endangered and endemic tree from the Brazilian Atlantic Forest Daniela B. Borges • Eduardo Mariano-Neto Fernanda A. Gaiotto
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Received: 15 July 2014 / Accepted: 29 July 2014 / Published online: 8 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Using next-generation sequencing, we developed and characterized microsatellite primers for Melanoxylon brauna, an important timber species endemic to the Atlantic forest. To characterize the primers, 32 young trees were sampled at one population. Of the 48 primers designed, 43 showed positive amplification and 38 were polymorphic. The combined probability of paternity exclusion was higher than 0.999, and the identity index was lower than 0.001, indicating that the microsatellite primers were suitable for various molecular studies such as paternity testing, gene flow, population structure, and evolution. We believe that the application of this tool in the biological model will contribute to the information on the history of the Atlantic forest, as well as improve strategies and conservation measures for this Brazilian diversity hotspot. Keywords Next-generation sequencing Atlantic forest SSR Conservation genetics Endangered species The tree species Melanoxylon brauna, popularly known as ‘‘brau´na’’, belongs to the Fabaceae family, subfamily
Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0288-3) contains supplementary material, which is available to authorized users. D. B. Borges F. A. Gaiotto (&) Laborato´rio de Marcadores Moleculares, Centro de Biotecnologia e Gene´tica, Universidade Estadual de Santa Cruz (UESC), Rodovia Jorge Amado km 16, Ilhe´us, BA 45662-900, Brazil e-mail: [email protected] E. Mariano-Neto Departamento de Botaˆnica, Instituto de Biologia, Universidade Federal da Bahia (UFBA), Salvador, Brazil
Caesalpinoideae. It is an endemic Atlantic forest tree that can reach a height of 40 m and occurs from Bahia to Sao Paulo in seasonal forests and rainforests. Its wood is of high density, dark, durable, and highly valuable. Based on these superior characteristics, this species was widely exploited and it is currently included in the Official List of Threatened Flora of Brazil (IBAMA 2008). To study population structure at the molecular level, we developed 43 species-specific microsatellite primers by next generation sequencing (NGS) (454 pyrosequencing technology, Ecogenics GmbH, Schlieren, Switzerland). To construct the genomic library, DNA was extracted from fresh leaves of M. brauna collected in Lenc¸o´is, Bahia, Brazil (12°270 5300 S, 41°180 3800 W) using the protocol described by Borges et al. (2012). The library was constructed using DNA enriched with (AC)n and (GA)n probes. Primers were tested on 32 trees, sampled from Una, Bahia, Brazil (15°200 3300 S, 39°020 1000 W). Forty-eight primers pairs were designed and synthesized, although five did not amplify. The remaining 43 primers sh
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