National Institute of Biological Resources, Environmental Research Complex, Incheon, Korea.
PLoS One. 2011;6(11):e26933. doi: 10.1371/journal.pone.0026933. Epub 2011 Nov 1.
Microsatellites, a special class of repetitive DNA sequence, have become one of the most popular genetic markers for population/conservation genetic studies. However, its application to endangered species has been impeded by high development costs, a lack of available sequences, and technical difficulties. The water deer Hydropotes inermis is the sole existing endangered species of the subfamily Capreolinae. Although population genetics studies are urgently required for conservation management, no species-specific microsatellite marker has been reported.
We adopted next-generation sequencing (NGS) to elucidate the microsatellite markers of Korean water deer and overcome these impediments on marker developments. We performed genotyping to determine the efficiency of this method as applied to population genetics.
We obtained 98 Mbp of nucleotide information from 260,467 sequence reads. A total of 20,101 di-/tri-nucleotide repeat motifs were identified; di-repeats were 5.9-fold more common than tri-repeats. CA and AAC/AAT repeats were the most frequent di- and tri-repeats, respectively. Of the 17,206 di-repeats, 12,471 microsatellite primer pairs were derived. PCR amplification of 400 primer pairs yielded 106 amplicons and 79 polymorphic markers from 20 individual Korean water deer. Polymorphic rates of the 79 new microsatellites varied from 2 to 11 alleles per locus (H(e): 0.050-0.880; H(o): 0.000-1.000), while those of known microsatellite markers transferred from cattle to Chinese water deer ranged from 4 to 6 alleles per locus (H(e): 0.279-0.714; H(o): 0.300-0.400).
Polymorphic microsatellite markers from Korean water deer were successfully identified using NGS without any prior sequence information and deposited into the public database. Thus, the methods described herein represent a rapid and low-cost way to investigate the population genetics of endangered/non-model species.
微卫星是一类特殊的重复 DNA 序列，已成为群体/保护遗传学研究中最流行的遗传标记之一。然而，由于开发成本高、可用序列缺乏和技术困难，其在濒危物种中的应用受到了阻碍。水鹿是唯一现存的小麂亚科濒危物种。尽管保护管理急需进行种群遗传学研究，但尚未报道任何物种特异性的微卫星标记。
我们采用下一代测序（NGS）阐明了韩国水鹿的微卫星标记，克服了标记开发方面的这些障碍。我们进行了基因分型，以确定该方法在种群遗传学中的应用效率。
我们从 260,467 条序列读取中获得了 98 Mbp 的核苷酸信息。总共鉴定出 20,101 个二核苷酸/三核苷酸重复基序；二重复比三重复常见 5.9 倍。CA和AAC/AAT重复分别是最常见的二重复和三重复。在 17,206 个二重复中，衍生出 12,471 对微卫星引物。从 20 只韩国水鹿个体中，PCR 扩增了 400 对引物，得到了 106 个扩增子和 79 个多态性标记。79 个新微卫星的多态性率在每个位点为 2-11 个等位基因（H(e)：0.050-0.880；H(o)：0.000-1.000），而从牛转移到中国水鹿的已知微卫星标记的多态性率在每个位点为 4-6 个等位基因（H(e)：0.279-0.714；H(o)：0.300-0.400）。
本研究无需任何先验序列信息，利用 NGS 成功鉴定了韩国水鹿的多态性微卫星标记，并将其存入公共数据库。因此，本文所述方法代表了一种快速且低成本的方法，可用于研究濒危/非模式物种的种群遗传学。
