Development of Microsatellite Markers for Viscum coloratum (Santalaceae) and Their Application to Wild Populations Author(s): Bo-Yun Kim, Han-Sol Park, Soonok Kim, and Young-Dong Kim Source: Applications in Plant Sciences, 5(1) Published By: Botanical Society of America DOI: http://dx.doi.org/10.3732/apps.1600102 URL: http://www.bioone.org/doi/full/10.3732/apps.1600102 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. 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Applications in Plant Sciences 2017 5(1): 1600102 Applications in Plant Sciences PRIMER NOTE DEVELOPMENT OF MICROSATELLITE MARKERS FOR VISCUM COLORATUM (SANTALACEAE) AND THEIR APPLICATION 1 TO WILD POPULATIONS BO-YUN KIM2, HAN-SOL PARK2, SOONOK KIM3, AND YOUNG-DONG KIM2,4 2Department of Life Science, Hallym University, Chuncheon 24252, Republic of Korea; and 3Biological and Genetic Resources Assessment Division, National Institute of Biological Resources, Incheon 22689, Republic of Korea • Premise of the study: Microsatellite primers were developed for Viscum coloratum (Santalaceae), a semiparasitic medicinal plant that is known for its anticancer properties. Due to excessive human harvesting and loss of suitable habitat of its populations, it has become a potentially threatened species requiring immediate conservation efforts. • Methods and Results: Based on transcriptome data for V. coloratum, 124 primer pairs were randomly selected for initial valida- tion, of which 19 yielded polymorphic microsatellite loci, with two to six alleles per locus. The usefulness of these markers was assessed for 60 individuals representing three populations of V. coloratum. Observed and expected heterozygosity values ranged from 0.033 to 0.833 and 0.032 to 0.672, respectively. Cross-species amplification for 19 loci in the related species V. album was conducted. • Conclusions: The 19 newly developed loci are expected to be useful for studying the population genetics and ecological conser- vation of V. coloratum. Key words: genetic diversity; medicinal plant; microsatellite; mistletoe; Santalaceae; Viscum coloratum. Mistletoes have been proposed to be a keystone resource in- diversity and population structure of V. coloratum should be im- fluencing biodiversity in forest ecosystems globally (Cooney mediately investigated for resource conservation. Despite the and Watson, 2008). The Korean mistletoe, Viscum coloratum ecological and medical importance of V. coloratum, no studies (Kom.) Nakai (Santalaceae), is distributed in many countries, have evaluated the genetic diversity in wild populations of this including Korea, Japan, China, and Russia (Qiu and Gilbert, species. 2003). Viscum L. species have lectins that are known for their Expressed sequence tags–simple sequence repeats (EST-SSRs) potential therapeutic, immunomodulatory, and anticancer prop- have proven valuable for their cross-transferability, facilitating erties (Lavastre et al., 2002; Lyu and Park, 2007). According to studies of population genetic diversity in many plant species previous studies, V. coloratum possesses similar cytotoxic and (Dikshit et al., 2015; Zhou et al., 2016). In this study, 19 poly- immunological activities as seen in European mistletoe, V. al- morphic microsatellite loci for V. coloratum were developed bum L. (Lee et al., 2009; Lyu and Park, 2010). Such uses have based on EST data obtained from Illumina paired-end sequenc- led to a great demand for these plants, resulting in the large-scale ing. The usefulness of these markers was assessed for 60 indi- harvesting of wild populations of V. coloratum. The increasing viduals representing three populations of V. coloratum in Korea, demand has raised concerns about its status as a potentially Japan, and China. Cross-species amplification was tested using threatened species. Recently, the environmental management of 20 individuals of V. album, a close relative of V. coloratum. mistletoes for conservation has become an international focus. For example, the International Union for Conservation of Na- ture (IUCN) has listed 19 species of mistletoe on the official METHODS AND RESULTS IUCN Red List of Threatened Species (International Union for Conservation of Nature, 2006). For this reason, the genetic We collected 60 individuals of V. coloratum from natural populations from three countries (Korea, Japan, and China), and the voucher specimens repre- senting each population were deposited in the Herbarium of the National Insti- 1 tute of Biological Resources (KB) and the Herbarium of Hallym University Manuscript received 27 August 2016; revision accepted 10 November (HHU), Republic of Korea (Appendix 1). To test cross-species amplification, 2016. we collected 20 individuals of V. album from a single population in Japan This research was supported by the grant “The Genetic and Genomic (Appendix 1). Whole genomic DNA was extracted from silica gel–dried leaf Evaluation of Indigenous Biological Resources” (NIBR201403202), tissue using the DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA). funded by the National Institute of Biological Resources, Republic of DNA concentrations were estimated using the NanoDrop 2000c (Thermo Korea. Fisher Scientific, Waltham, Massachusetts, USA), and samples were stored 4 Author for correspondence: [email protected] at −20°C. For RNA library construction, total RNA was extracted from the leaf of a doi:10.3732/apps.1600102 single individual plant collected from Korea (voucher no.: GEIBGR0000298682; Applications in Plant Sciences 2017 5(1): 1600102; http://www.bioone.org/loi/apps © 2017 Kim et al. Published by the Botanical Society of America. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-NC-SA 4.0), which permits unrestricted noncommercial use and redistribution provided that the original author and source are credited and the new work is distributed under the same license as the original. 1 of 4 Applications in Plant Sciences 2017 5(1): 1600102 Kim et al.—Viscum microsatellite markers doi:10.3732/apps.1600102 Appendix 1). Total RNA quality and quantity were verified using the NanoDrop Microsatellites were detected using the Perl script MIcroSAtellite (MISA) 2000c (Thermo Fisher Scientific) and Bioanalyzer 2100 (Agilent Technologies, identification tool (Thiel et al., 2003) with thresholds of 10 repeat units for Santa Clara, California, USA). We constructed Illumina-compatible transcrip- mononucleotides, six for dinucleotides, and five for tri-, tetra-, penta-, and hexa- tome libraries using a TruSeq RNA Library Preparation Kit version 2 (Illumina, nucleotides. MISA identified 15,562 microsatellite sequences, of which 124 loci San Diego, California, USA), according to the manufacturer’s instructions. In were selected for further testing (based on the above criteria) in 60 individuals of brief, mRNA was purified from total RNA by polyA selection, and was then V. coloratum from three countries (Appendix 1). Primers were designed using chemically fragmented and converted into single-stranded cDNA with random Primer3 (Rozen and Skaletsky, 1999) to flank the microsatellite-rich regions hexamer-primed reverse transcription. A second cDNA strand was generated to with a minimum of six repeats. create double-stranded cDNA for TruSeq library construction. The short double- PCRs were performed in a total volume of 25 μL containing 10× Ex Taq stranded cDNA fragments were then connected using sequencing adapters. buffer (TaKaRa Bio Inc., Otsu, Shiga, Japan) 2.5 μL, 2.5 mM dNTPs 2 μL, 0.01 Finally, RNA libraries were built by PCR amplification. The RNA libraries μM forward primers, 0.01 μM reverse primers, 5 units TaKaRa Ex Taq (TaKaRa were quantified using real-time PCR (qPCR), according to the qPCR Quan- Bio Inc.) 0.1 μL, 5–10 ng template DNA, and distilled water up to the final tification Protocol Guide (Illumina), and qualified using an Agilent 2200 volume. Reactions were performed in a GeneAmp PCR System 9700 thermo- Bioanalyzer. cycler (Applied Biosystems, Carlsbad, California, USA) programmed with an Paired-end 150-bp sequencing of V. coloratum was conducted on the Illu- initial denaturation step at 98°C for 5 min; followed by 30 cycles of denatur- mina HiSeq 2000 platform. All sequence information has been deposited in the ation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for National Center for Biotechnology Information (NCBI) Sequence Read Ar- 1.5 min; and a final extension step at 72°C for 10 min. Fluorescently labeled PCR chive (Bioproject no. SRP092226). Adapter/quality trimming was performed products were analyzed using an ABI 3730XL sequencer with the GeneScan using Trimmomatic 0.32 (Bolger