Conservation Genetics Resources (2020) 12:585–587 https://doi.org/10.1007/s12686-020-01162-8 TECHNICAL NOTE Development of nuclear SNP markers for Mahogany (Swietenia spp.) Birte Pakull1 · Lasse Schindler1 · Malte Mader1 · Birgit Kersten1 · Celine Blanc‑Jolivet1 · Maike Paulini1 · Maristerra R. Lemes2 · Sheila E. Ward3 · Carlos M. Navarro4 · Stephen Cavers5,8 · Alexandre M. Sebbenn6 · Omar di Dio6 · Erwan Guichoux7 · Bernd Degen1 Received: 6 April 2020 / Accepted: 23 July 2020 / Published online: 12 August 2020 © The Author(s) 2020 Abstract Swietenia species are the most valuable American tropical timbers and have been heavily overexploited for decades. The three species are listed as either vulnerable or endangered by IUCN and are included on Appendix II of CITES, yet illegal exploitation continues. Here, we used restriction associated DNA sequencing to develop a new set of 120 SNP markers for Swietenia sp., suitable for MassARRAY®iPLEX™ genotyping. These markers can be used for population genetic studies and timber tracking purposes. Keywords SNPs · Mahogany · Swietenia spp. · MassARRAY®iPLEX™ The genus Swietenia includes the species: Swietenia mahag- commercially because of past overexploitation, S. macro- oni (L.) Jacq. (Small-leaved mahogany, native to Florida phylla is now the most valuable and economically important and the Caribbean islands), Swietenia macrophylla King. American tropical timber (Louppe et al. 2008). Swietenia (Big-leaved mahogany, native to Central and South Amer- wood is used for high-class furniture, boat building, musical ica) and Swietenia humilis Zucc. (Pacifc Coast mahogany, instruments etc. All three mahogany species are listed on native to the relatively dry Central American Pacifc coast) CITES (Convention on International Trade in Endangered (Schütt et al. 2014). While S. mahagoni is no longer traded Species, Appendix II) and on the IUCN red list, where S. macrophylla and S. humilis are listed as vulnerable and S. mahagoni Electronic supplementary material The online version of this is classifed as endangered. article (https ://doi.org/10.1007/s1268 6-020-01162 -8) contains Illegal logging is a major threat to the sustainable use of supplementary material, which is available to authorized users. natural forests. Laws like the European Union Timber Regu- lation and an amendment of the Lacey Act in the US prohibit * Birte Pakull [email protected] the trade of illegally sourced timber. However, existing tim- ber-tracking/chain-of-custody systems are based mainly on 1 Thünen Institute of Forest Genetics, Sieker Landstrasse 2, electronic tags or paper-based documentation, and are vul- 22927 Grosshansdorf, Germany nerable to falsifcation. Therefore, eforts to establish genetic 2 Laboratório de Genética e Biologia Reprodutiva de Plantas, methods for timber tracking are being undertaken. Several Instituto Nacional de Pesquisas da Amazônia, Av André academic studies documenting population genetic structure Araújo 2936, Manaus, AM 69057-375, Brazil in Swietenia species have been completed, based on the anal- 3 Mahogany for the Future Inc., San Juan, PR 00928, USA ysis of nuclear and chloroplast microsatellite (SSR) mark- 4 Universidad Nacional, Calle 9, Avenidas 0 y 9, Heredia, ers (e.g. Degen et al. 2013; Lemes et al. 2010; Lemes et al. Costa Rica 2003; Novick et al. 2003). However, genotyping of SSR 5 UK Centre for Ecology & Hydrology, Midlothian, Penicuik, loci from timber material can be difcult as DNA extracts Scotland EH6 0QB, UK are often low quality, which impedes the amplifcation of 6 Instituto Florestal de São Paulo, CP 1322, São Paulo, relatively long SSR-fragments (typically 100–300 bp). Con- SP 01059-970, Brazil sequently, single nucleotide polymorphism (SNP) markers 7 University of Bordeaux, INRAE, BIOGECO, 33610 Cestas, have become the marker of choice for timber forensics, as France they can be characterized in much shorter fragments, are 8 Bush Estate, Midlothian, Penicuik, Scotland EH6 0QB, UK Vol.:(0123456789)1 3 586 Conservation Genetics Resources (2020) 12:585–587 simple to standardize among laboratories, and are fast and or contaminated negative control were removed from the low-cost to develop in large numbers (Blanc-Jolivet et al. dataset, leaving a total of 384 loci for further analysis. Indi- 2017). SNP sets have been developed for timber tracking viduals were then grouped according to: (1) species (all purposes in other commercially important tropical tree spe- individuals) and (2) country of origin (individuals of each cies, e.g., Hymenaea spp. (Chaves et al. 2019), Dipteryx species separately). For each grouping we calculated: the spp. (Honorio Coronado et al. 2019), Cedrela spp. (Paredes- percentage of analyzable individuals per marker, the fxation Villanueva et al. 2019), Jacaranda copaia (Sebbenn et al. index (FIS) as an indicator of potential null alleles or allele 2019), Carapa spp. (Tysklind et al. 2019), Entandrophragma fxation within a group (Nei and Chesser 1983), the efective cylindricum (Blanc-Jolivet et al. 2018), Handroanthus spp. number of alleles (A e), the genetic diferentiation (δ) among (Meyer-Sand et al. 2018) and Milicia spp. (Blanc-Jolivet groups (Gregorius 1987), the correlation between genetic et al. 2017). and geographic distance (only when grouped according to Here, we describe a new set of 120 SNPs for Swietenia country of origin), and population fxation index FST (Nei spp., which can be used for population genetic studies and and Chesser 1983), using GDANT (Degen, unpublished, timber tracking purposes. Suppl. 2). A fnal marker set of 120 SNPs was determined, Tissues (predominantly leaves) from S. macrophylla, prioritizing markers with high rates of amplifcation, high S. humilis and S. mahagoni were harvested in Central and diferentiation power among groups, and—if available— South American countries (Brazil, Belize, Bolivia, Costa high correlation between genetic and geographic distance. Rica, El Salvador, Guatemala, Honduras, Mexico, Nicara- The list of the 120 SNP loci included in the fnal marker set gua, and Panama). Samples were dried with silica gel. DNA is in Suppl. 3. isolation from dried cambium and leaf material was car- ried out according to Dumolin et al. (1995). DNA isolation from wood material was carried out according to Lowe et al. Acknowledgements Open Access funding provided by Projekt DEAL. This research was supported by the German Federal Ministry of Food (2015). SNP discovery was based on restriction-associated and Agriculture (“Large scale project on genetic timber verifcation”, DNA sequencing (RADseq, Miller et al. 2007). RADseq was project No. 28I-001-01). Genotyping was performed at the Genome carried out by Floragenex (Portland, USA) using genomic Transcriptome Facility of Bordeaux Genomic and Sequencing Facil- DNA from fve individuals of S. macrophylla originating ity of Bordeaux (grants from Investissements d’Avenir, Convention attributive d’aide EquipEx Xyloforest: ANR-10-EQPX-16-01grants from Bolivia, Brazil, Costa Rica, Honduras and Mexico. from the Conseil Regional d’Aquitaine no. 20030304002FA and Libraries were prepared with SbfI and sequenced using 20040305003FA, the European Union, FEDER no. 2003227 and Inves- 2 × 100 bp paired-end Illumina HiSeq. The Brazilian sam- tissements d’avenir, no. ANR-10-EQPX-16-01 and CEBA: ANR-10- ple was used for reference assembly. A total of 2450 SNPs LABX-25-01 to the UMR EcoFoG), with the help of Marie Massot. (variant call format 4.1) were identifed on 1011 reference Open Access This article is licensed under a Creative Commons Attri- contigs (stringent mapping conditions). An initial set of 480 bution 4.0 International License, which permits use, sharing, adapta- SNP markers was compiled based on the results of the RAD- tion, distribution and reproduction in any medium or format, as long seq approach. Selection criteria were: strictly no other SNP as you give appropriate credit to the original author(s) and the source, present within at least 50 bp on both sides of a selected SNP, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are location on diferent scafolds, sufcient sequence coverage included in the article’s Creative Commons licence, unless indicated and diferent patterns of allele distribution among the difer- otherwise in a credit line to the material. If material is not included in ent individuals used for sequencing. The 480 selected SNPs the article’s Creative Commons licence and your intended use is not were used for MassARRAY®iPLEX™ (Agena Biosciences, permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a Hamburg, Germany) genotyping. Alleles were called using copy of this licence, visit http://creativeco mmons .org/licen ses/by/4.0/ . Typer Viewer v.4.0.24.71 (Agena Biosciences). The screen- ing included 94 samples (Suppl. 1), representing all three species (S. macrophylla: 69 individuals, S. humilis: 11 indi- viduals, S. mahagoni: 10 individuals), two wood samples References and two individuals of the related species Carapa guianen- sis and Carapa procera. The individuals originated from at Blanc-Jolivet C, Kersten B, Daïnou K, Hardy O, Guichoux E, Del- least 13 diferent countries (Brazil, Belize, Bolivia, Costa camp A, Degen B (2017) Development of nuclear SNP markers for genetic tracking of Iroko, Miliciaexcelsa and Miliciaregia. Rica, Dominican Republic, El Salvador,