Isolation and characterization of microsatellite markers for the neotropical tree, Pachira quinata (Malvaceae)
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MICROSATELLITE LETTERS
Isolation and characterization of microsatellite markers for the neotropical tree, Pachira quinata (Malvaceae) Paul D. Rymer • Stephen Harris • David Boshier
Received: 4 November 2013 / Accepted: 25 November 2013 Ó Springer Science+Business Media Dordrecht 2014
Abstract Twelve microsatellite loci were developed from a genomic library enriched for SSRs of Pachira quniata, a canopy tree species of dry and some humid forests in the neotropics (Central and northern South America). Genotyping of 30 trees from one population on the Pacific coast of Nicaragua was successfully performed. The loci were across amplified in related species. These microsatellites will be used to compare regional population structuring and mating patterns in fragmented landscapes. Keywords Malvaceae Bombacaceae SSR markers Neotropical forest
Pachira quinata (Jacq.) (Alverson 1994) (syn: Bombacopsis quinata) is a medium to large-size deciduous tree, highly valued for its timber within its native range from Honduras in Central America, to Colombia and Venezuela in South America (listed as vulnerable by IUCN). It is hermaphroditic, largely self-incompatible, and principally bat pollinated (Sandiford 1998). We aimed to characterize highly polymorphic microsatellite (SSR) markers to be used in future studies on the distribution and abundance of neutral genetic variation, and the estimation of mating patterns in natural populations.
P. D. Rymer (&) Hawkesbury Institute for the Environment, University of Western Sydney, Bourke Street, Richmond, NSW 2753, Australia e-mail: [email protected] P. D. Rymer S. Harris D. Boshier Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
Seed from one P. quinata tree collected from Nambaras, Nicaragua (12°100 N, 85°380 W) was extracted using a QIAGEN DNeasy plant mini kit to obtain total genomic DNA. The microsatellite-enriched library was constructed using the protocol described by Glenn and Schable (2005), with minor modifications (McPherson et al. 2008). Fifty clones enriched with the mixed probes and two hundred and twenty AG-rich clones were ethanol purified and sequenced using universal M13 primers and BigDye terminator version 3.1 (AppliedBiosystems). Fifty-one of these sequences contained SSRs (see Appendix for sequences), 27 of which were suitable to design locus-specific primers based on primer3 software and NETPRIMER (Premier biosoft). Twenty-two were synthesized and tested as unlabeled products, 15 were selected for genotyping analysis being labeled with fluorescent dyes (6FAM, PET, VIC and NED; Applied Biosystems) and, of these, 12 loci were informative (Table 1). DNA extracts from 30 individuals from a remnant forest in Nambaras (Nicaragua) were then genotyped. PCR amplifications were performed in an ABI Thermal cycler. Amplification conditions were 94 °C for 3 min; 30 cycles 94 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s; and a final extension at 72 °C for 10 min. Yorkshire Biosciences TAQ polymerase, 109 buffer and MgCl2, Ne
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