Sixteen Polymorphic Microsatellite Markers for a Federally Threatened Species, Hexastylis naniflora (Aristolochiaceae), and Co-Occurring Congeners Author(s): Jacqueline W. Hamstead, Brandon L. Snider, Robyn Oaks, Evan Fitzgerald, Jason Woodward, Alyssa Teat, Nikolai M. Hay, Matt C. Estep, and Zack E. Murrell Source: Applications in Plant Sciences, 3(7) Published By: Botanical Society of America DOI: http://dx.doi.org/10.3732/apps.1500033 URL: http://www.bioone.org/doi/full/10.3732/apps.1500033 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. ApApplicatitionsons Applications in Plant Sciences 2015 3 ( 7 ): 1500033 inin PlPlant ScienSciencesces P RIMER NOTE S IXTEEN POLYMORPHIC MICROSATELLITE MARKERS FOR A FEDERALLY THREATENED SPECIES, H EXASTYLIS NANIFLORA (ARISTOLOCHIACEAE), AND CO-OCCURRING CONGENERS 1 J ACQUELINE W . H AMSTEAD 2 , B RANDON L . S NIDER 2 , R OBYN O AKS 2 , E VA N F ITZGERALD 2 , J ASON W OODWARD 2 , A LYSSA T EAT 2 , N IKOLAI M . H AY 2 , M ATT C. ESTEP 2,3 , AND Z ACK E. MURRELL 2 2 Department of Biology, Appalachian State University, 572 Rivers Street, Boone, North Carolina 28608 USA • Premise of the study: Twenty microsatellite loci were developed for the federally threatened species Hexastylis nanifl ora (Aris- tolochiaceae) to examine genetic diversity and to distinguish this species from co-occurring congeners, H. heterophylla and H. minor . • Methods and Results: Next-generation sequencing approaches were used to identify microsatellite loci and design primers. One hundred fi fty-two primer pairs were screened for repeatability, and 20 of these were further characterized for polymorphism. In H. nanifl ora , the number of alleles identifi ed for polymorphic loci ranged from two to 23 (mean ~8.8), with a mean hetero- zygosity of 0.39. • Conclusions: These 16 polymorphic primers for H. nanifl ora will be useful tools in species identifi cation and quantifying ge- netic diversity within the genus. Key words: Aristolochiaceae; Asarum ; Hexastylis ; Hexastylis nanifl ora ; hybrid; microsatellite markers. The segregate genus Hexastylis Raf. (Aristolochiaceae), of- identify evolutionarily signifi cant units to aid in the manage- ten included in Asarum L., is an enigmatic group of 12 species ment of these species. These markers have the potential to iden- distributed in the southeastern United States ( Blomquist, 1957 ; tify species and hybrids in their vegetative state, allowing land Niedenberger, 2010 ). Hexastylis has been segregated based upon managers to evaluate population value and management strate- its entirely North American distribution, karyotype ( Sugawara, gies throughout the year, instead of only during the short fl ow- 1981 ; Soltis, 1984 ), pollen morphology ( Niedenberger, 2010 ), ering period. and several characteristics of fl ower morphology ( Gaddy, 1987 ). Multiple species complexes have been identifi ed in this genus and this study focuses on the H. heterophylla complex, METHODS AND RESULTS containing H. heterophylla (Ashe) Small, H. minor (Ashe) H. L. Blomq., and H. nanifl ora H. L. Blomq. The species in this Leaf tissue was collected and preserved on silica gel from plants at 15 sites complex are sympatric over portions of their ranges. Vegetative in North and South Carolina (Appendix 1). Tissue samples from one plant of H. characters have limited taxonomic value, leaving ephemeral nanifl ora and one plant of H. heterophylla (selected from geographic ranges fl oral morphology as the only diagnosable fi eld character for where the species do not overlap and confi dently identifi ed using fl ower mate- identifi cation. Previous studies have recognized intermediate rial) were sent to the Cornell University Evolutionary Genetics Core Facility where total DNA was extracted using a QIAGEN Plant Mini Kit (QIAGEN, fl oral morphologies in some populations, leading some to ques- Valencia, California, USA). Restriction enzymes Alu I, Hpy 166II, and Rsa I tion the validity of species circumscriptions. This is particularly (New England Biolabs, Ipswich, Massachusetts, USA) were used to digest the problematic in H. nanifl ora , where land managers and conser- DNA, which was then ligated to an Illumina Y-adapter (Illumina, San Diego, vation biologists are tasked with protection of this federally California, USA) using T4 DNA ligase. The DNA fragments were then hybridized ′ threatened species. to 3 biotinylated oligonucleotide repeat probes: (GT)8 , (TC)9.5 , (TTTTG)4.2 , Through funding from the North Carolina Department of (TTTTC)4.6 , (TTC)7 , (GTA)8.33 , (GTG)4.67 , (TCC)5 , (GTT)6.33 , (TTTC)6 , (GATA)7 , (TTAC)6.75 , (GATG)4.25 , (TTTG)5.25 , (TTTTG)4.2 , (TTTTC)4.6 . Enriched frag- Transportation, 16 polymorphic microsatellite markers were ments were then captured by streptavidin-coated magnetic beads (New England developed to help distinguish H. nanifl ora from H. minor and Biolabs) and PCR amplifi ed. Agarose gel and a Qubit 2.0 Fluorometer (Life H. heterophylla , to address questions of hybridization, and to Technologies, Grand Island, New York, USA) were used to analyze the PCR product, and fragments 300–600 bp were recovered with AMPure beads (Beck- 1 man Coulter , Brea, California, USA). Samples were then moved to Cornell Life Manuscript received 25 March 2015; revision accepted 27 April 2015. Sciences Sequencing and Genotyping Facility for sequencing on an Illumina We acknowledge Margaret Roberts, Taylor Jenson, and George Godsmark MiSeq. Raw sequence reads were then assembled using SeqMan NGen (v.11, for efforts that were vital to the collection of plant material. We also thank Lasergene Genomics Suite; DNASTAR , Madison, Wisconsin, USA). Contigs the Department of Biology at Appalachian State University, the North containing microsatellite repeats were identifi ed using MSATCOMMANDER Carolina Department of Transportation, and the North Carolina Natural version 1.0.3 ( Faircloth, 2008 ), and possible primer pairs were identifi ed. Heritage Program for funding and support. One hundred fi fty-two primer pairs were selected to screen for amplifi cation 3 Author for correspondence: [email protected] in eight individuals: six H. nanifl ora , one H. heterophylla , and one H. minor. PCR amplifi cations were prepared in a 10- μ L reaction consisting of GoTaq μ μ doi:10.3732/apps.1500033 Flexi Buffer, 2.5 mM MgCl2 , 800 M dNTPs, 0.5 M of each primer, 0.5 units Applications in Plant Sciences 2015 3 ( 7 ): 1500033; http://www.bioone.org/loi/apps © 2015 Hamstead et al. Published by the Botanical Society of America. This work is licensed under a Creative Commons Attribution License (CC-BY-NC-SA). 1 of 3 Applications in Plant Sciences 2015 3 ( 7 ): 1500033 Hamstead et al.— Hexastylis nanifl ora microsatellites doi:10.3732/apps.1500033 of GoTaq Flexi DNA Polymerase, and ~20 ng of DNA (Promega, Madison, Sixteen of the primer pairs tested were polymorphic, with the number of Wisconsin, USA). PCR was completed using a touchdown thermal cycling pro- alleles ranging from two to 23 (mean ~8.8) in H. nanifl ora , two to nine (mean gram on a Techne TC-5000 Thermal Cycler (Bibby Scientifi c Limited, Stone, ~4.9) in H. minor , and one to 14 (mean ~6.1) in H. heterophylla ( Table 2 ). Ex- Staffordshire, United Kingdom) encompassing a 13 ° C span of annealing tem- cessive homozygosity was identifi ed at several of the loci in all three species, peratures from 68 ° C to 55 ° C. Initial denaturation was at 94 ° C for 5 min, 13 and locus Hn00567 was monomorphic in H. heterophylla . A total of 52 private cycles at 94 ° C for 45 s, touchdown for 2 min, and 72 ° C for 1 min; followed by alleles were identifi ed in one of the three species, mostly at low frequencies 24 cycles at 94 ° C for 45 s, 55 ° C for 1 min, and 72 ° C for 1 min; followed by a (<0.05). Three of these private alleles in H. nanifl ora (Hn7116 [422 bp], fi nal extension at 72 ° C for 5 min. The PCR products were examined on a 1% Hn01135 [300 bp], and Hn00304 [179 bp]), one in H. minor (Hn00252 agarose gel and scored for presence or absence of an appropriately sized PCR [224 bp]), and one in H. heterophylla (Hn00002 [297 bp]) were identifi ed with product. Twenty primer pairs produced repeatable results across all three spe- a frequency greater than 10%, and these can be diagnostic in species identifi ca- cies ( Table 1 ) . These were further screened for polymorphism on a total of 68 tion when morphological characters are unavailable. individuals, including 44 H. nanifl ora , 10 H. minor , and 14 H. heterophylla (Appendix 1). Polymorphism screening PCR reaction conditions were the same as above, CONCLUSIONS except the forward primer concentration was reduced to 0.25 μ M, and 0.25 μ M of an M13 primer (5 ′ -CACGACGTTGTAAAACGAC-3 ′ ), labeled with FAM, VIC, NED, or PET (Life Technologies), was added to the reaction. PCR prod- Sixteen polymorphic microsatellite markers were devel- ucts labeled with different fl uorescent dyes were then pseudo-multiplexed, and oped for H. nanifl ora , and these primers also amplify in two 2 μ L of the combined reactions were submitted for genotyping on an ABI 3730 other species of Hexastylis ( H.