Molecular Ecology Notes (2006) 6, 72–74 doi: 10.1111/j.1471-8286.2006.01141.x © 2006 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Characterization of six polymorphic microsatellite loci isolated from Hymenocallis coronaria (J. LeConte) Kunth (Amaryllidaceae) S. H. MARKWITH* and M. J. SCANLON† Departments of *Geography and † Plant Biology, University of Georgia, Athens, Georgia 30602, USA Abstract Microsatellite loci were isolated from the allotetraploid aquatic plant Hymenocallis coro- naria. A repeat-enriched genomic library was constructed and primer pairs designed, resulting in six polymorphic loci. A total of 230 individuals were genotyped, and allelic richness per locus ranged from three to 11, while observed heterozygosity ranged from 0.017 to 0.570. Some amplified products were excised from agarose gel and sequenced to confirm primer specificity and mutation model. These are the first microsatellite markers developed for any member of this genus, and cross-amplification was successful with the only other member of the genus tested and with a member of the related genus Zephyranthes. Keywords: Cahaba lily, macrophyte, Shoals spider lily, SSR primers Received 16 June 2005; revision accepted 18 July 2005 Hymenocallis coronaria, commonly known as the Shoals spider lily or Cahaba lily, is an emergent macrophyte found in streams of the piedmont and ridge and valley physiographical provinces of Georgia, South Carolina, and Alabama. It is restricted to rocky shoals, where its bulbs and roots find hold within rock crevices and accumulated sediments (Davenport 1996). There are currently only 65 known natural populations found in a disjunct distribution within 10 major drainage basins. The relative rarity of H. coronaria and continued threats to its populations, such as water resource development, have prompted various forms of state-level protection across the species’ range. Hymenocallis coronaria is also an allotetraploid (2 n = 44) in a genus that recently underwent, or is continuing through a period of rapid speciation via hybridization and major chromosomal rearrangements ( Joye & Smith 1993). Here we characterize six polymorphic di- and trinucleotide simple sequence repeat (SSR) loci for the population genetic analysis of H. coronaria which represent the first molecular genetic markers developed specifically for a member of this genus. Genomic DNA was isolated from the leaf tissue of H. coronaria using a urea-based protocol (Chen & Dellaporta 1994). A genomic DNA library enriched for di- and trinu- cleotide repeat sequences was created following the repeat enrichment procedure of Glenn & Schable (2005) with minor variation. Genomic DNA was cleaved using Rsa I and Xmn I (New England BioLabs) restriction enzymes and double-stranded linkers were ligated to the DNA fragments. Polymerase chain reaction (PCR) was performed using a PTC-100 Programmable Thermal Controller (MJ Research) on 5 μ L of linker-ligated DNA in a reaction containing 3 μ L of single-stranded linker primer, 12.5 μ L of Taq PCR Master Mix (QIAGEN) and 4.5 μ L H 2 O; with a 95 °C 2-min dena- turation cycle; then 25 cycles at 95 ° C for 20 s, 52 ° C for 20 s, 72 ° C for 1.5 min; and a hold cycle at 15 ° C. A mix of 5 ′ - biotinylated microsatellite probes containing (ACA) 10 , (TA) 15 , (CA) 15 , (GA) 15 , (AGA) 10 and (TGA) 10 sequences was hybridized to the PCR product and affinity-captured on Dynabeads M-280 Streptavidin (Dynal Biotech) with a magnetic particle collecting unit. The captured fragments were purified and a 5- μ L aliquot was used in a PCR con- taining the same reagents as previous; the PCR cycle was changed only by adding a 30-min 72 ° C step prior to the hold cycle. PCR product was ligated into pCR II-TOPO vector plasmid (Invitrogen) and transformed into Escherichia coli OneShot TOP10 chemically competent cells (Invitro- gen) according to manufacturer’s protocols. Selected bacterial colonies were harvested, the plasmids purified and iso- Correspondence: S. H. Markwith, Fax: 706-542-2388; E-mail: markwith@uga.edu