Novel microsatellite markers for Distylium lepidotum (Hamamelidaceae) endemic to the Ogasawara Islands

BMC Research Notes Sugai and Setsuko BMC Res Notes (2016) 9:332 DOI 10.1186/s13104-016-2137-9 Open Access SHORT REPORT Novel microsatellite markers for Distylium lepidotum (Hamamelidaceae) endemic to the Ogasawara Islands Kyoko Sugai1* and Suzuki Setsuko2 Abstract Background: Distylium lepidotum is a small tree endemic to the Ogasawara Islands located in the northwestern Pacific Ocean. This species is a sole food for an endemic locust, Boninoxya anijimensis. Here, we developed microsatellite markers to investigate genetic diversity and genetic structure and to avoid a genetic disturbance after transplantation to restore the Ogasawara Islands ecosystem. Results: Microsatellite markers with perfect dinucleotide repeats were developed using the next-generation sequencing Illumina MiSeq Desktop Sequencer. Thirty-two primer pairs were characterized in two D. lepidotum populations on Chichijima and Hahajima Islands of the Ogasawara Islands. The number of alleles for the markers ranged from three to 23 per locus in the two populations. Expected heterozygosity per locus in each population ranged from 0.156 to 0.940 and 0.368 to 0.845, respectively. Conclusions: These microsatellite markers will be useful for future population genetics studies of D. lepidotum and provide a basis for conservation management of the Ogasawara Islands. Keywords: Distylium lepidotum, Next-generation sequencing, Ogasawara Islands, Population genetics, Simple sequence repeat Findings Background Microsatellite markers, or simple sequence repeats, are widely applicable as DNA-based markers for population genetics studies. Moreover, their cost-effective development has been increasingly facilitated by applying nextgeneration sequencing (NGS) technologies [20]. Distylium lepidotum Nakai (Hamamelidaceae) is a small tree endemic to the oceanic Ogasawara Islands in the northwestern Pacific Ocean. The species is the dominant tree in the Distylium–Pouteria dry scrub [18], which is inhabited by Boninoxya anijimensis Ishikawa, a locust recorded as a new genus and species [8]. The locust utilizes D. lepidotum as the sole food, i.e., it is monophagous [8, 9]. Although it is only distributed *Correspondence: 1 Laboratory of Wildlife Ecology, Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305‑8687, Japan Full list of author information is available at the end of the article on Anijima Island of the Ogasawara Islands, it has been exposed to alien predatory species such as Anolis carolinensis. Conservation/benign introduction measures of B. anijimensis are needed on the Ogasawara Islands, except Anijima Island, to protect the B. anijimensis populations. As D. lepidotum is an essential food source, it may be possible to transplant the species. Therefore, it is important to reveal the genetic structure of the species to minimize any genetic disturbance due to the transplant. Here, we developed microsatellite markers to investigate the genetic diversity and structure in D. lepidotum. Methods Microsatellite markers were developed for D. lepidotum using an Illumina MiSeq Desktop Sequencer (Illumina, San Diego, CA, USA). Total genomic DNA was extracted from one silica-gel dried D. lepidotum leaf sample collected from Chibusayama (26°39′17.4″N 142°10′03.6″E) on Hahajima Island of the Ogasawara Islands using a © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sugai and Setsuko BMC Res Notes (2016) 9:332 DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). A shotgun library was prepared using the Nextera DNA Sample Preparation Kit v2 (Illumina), and the raw de novo sequencing data were obtained using the MiSeq Reagent Kit v2 (500 cycles) (Illumina). The raw reads were divided into each index, extra sequences (adapters and indices) were trimmed, and FASTAQ files were generated using the MiSeq Reporter v.2.5.1 (Illumina). The paired-end reads were merged using PEAR 0.9.6 [21] with default parameter settings. After the paired-end assembly, the low quality reads (<95 % with Phred quality score of 30) were removed using the script fastq_quality_filter included in the FASTX-Toolkit v.0.0.14 [7]. The resulting FASTQ files were converted to FASTA format using the ShortRead package [12]. A total of 1734,031 contigs with an average length of 241 bp were obtained. The microsatellites were identified and the primer pairs were designed with QDD2.1 [11]. A total of 41,367 unique sequences containing pure/compound microsatellite regions (2–6 nucleotide motifs with >5 repeats) and primer-designable flanking regions were selected. The primer pairs were designed with Primer3 [17] and implemented in QDD2.1 using the following criteria: (1) polymerase chain reaction (PCR) product size of 90–500 bp and (2) primer lengths of 20–27 bp, melting temperature of 57–63° C, and GC content of 20–80 %. Finally, 18,239 microsatellite primer pairs were designed using Primer3. Amplification and polymorphism were confirmed in 48 selected primer pairs after considering the microsatellites (one single dinucleotide motif with more than ten repetitions), design type (“A” or “B” in QDD2.1), and PCR product size to apply multiplex amplification (Table 1). Four universal primers with different fluorescent tags designed by Blacket et al. [1] were prepared, and the 5′ end of each forward primer was attached to the same sequence as a tail. In addition, as the 5′ end sequences of each reverse primer became 5′-GTTT-3′, a PIG-tail (5′-GTTT-3′, 5′-GTT-3′, 5′-GT-3′, or 5′-G-3′) was added to reduce stuttering due to inconsistent addition of adenine by Taq DNA polymerase [2]. PCR amplification was performed using the QIAGEN Multiplex PCR Kit. Multiplex PCRs were performed for each of the four primer pair sets using the following thermal cycle conditions: initial denaturation for 15 min at 95° C, 35 cycles of denaturation for 30 s at 95° C, Page 2 of 6 annealing for 1.5 min at 57° C, extension for 1 min at 72° C, and final extension for 30 min at 60° C. The PCR products were separated by capillary electrophoresis on an ABI3130 Genetic Analyzer (Life Technologies, Waltham, MA, USA) with the GeneScan 600 LIZ Size Standard (Life Technologies). The fragments were sized using GeneMapper 4.0 (Life Technologies). We finally tested two populations from Chichijima and Hahajima Islands in the central part of the Ogasawara Islands to evaluate the allelic polymorphisms: 24 individuals from Asahiyama (27°05′ (...truncated)