Development of 20 microsatellite markers for the endangered Japanese golden eagle (Aquila
chrysaetos japonica) using next generation sequencing
Yu Sato, Hideyuki Ito, Manabu Onuma, Taku Maeda, Rob Ogden, Miho Inoue-Murayama*
Y. Sato, H. Ito, R. Ogden, M. Inoue-Murayama
Wildlife Research Center of Kyoto University, Kyoto 606-8203, Japan.
M. Onuma, M. Inoue-Murayama
National Institute for Environmental Studies, Tsukuba 305-8506, Japan.
T. Maeda
Research Institute for Environmental Science and Public Health of Iwate Prefecture, Morioka 0200857, Japan.
R. Ogden
Royal Zoological Society of Scotland, Edinburgh EH12 6TS, United Kingdom.
*Corresponding author. Tel.: +81-75-771-4375;
Fax: +81-75-771-4394
Email: mmurayama@wrc.kyoto-u.ac.jp
Abstract
The Japanese golden eagle (Aquila chrysaetos japonica) is an endangered species in Japan. Genetic
studies are important for conservation, but currently available genetic markers are insufficient for
precise genetic studies with non-invasive samples. Therefore we developed twenty species-specific
microsatellite markers using next generation sequencing and tested them against a sample of
Japanese golden eagles. The number of alleles per locus ranged from two to seven (mean 3.7). The
range of observed (Ho) and expected heterozygosities (He) was 0.182–0.727 (mean 0.481) and
0.313–0.744 (mean 0.542), respectively.
Keywords Japanese golden eagle; Next generation sequencing; Microsatellite
The golden eagle (Aquila chrysaetos) is distributed widely in the northern hemisphere and is
categorized as Least Concern by the IUCN. However, the Japanese subspecies (A. c. japonica) is
distributed only in North-East Asia, and the population size in Japan is estimated to be around 500
(SRGE 2014). In Japan, genetic structure and diversity have not been investigated. Since hunting and
capture of wild golden eagles is prohibited, genetic analysis relies on non-invasive samples, often of
poor DNA quality. The aim of this study was to isolate novel microsatellite markers within the target
subspecies to maximize specificity and reduce genotyping error in such samples.
DNA was extracted from the blood of a female golden eagle from Akita Omoriyama Zoo using
QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA). DNA sequencing was conducted
on an Ion Torrent PGMTM sequencer (Life Technologies). Among the 5,222,991 reads obtained, 2,424
reads containing at least 8 di-, 7 tri- or 7 tetra- nucleotide repeats were identified by
Msatcommander (Faircloth 2008). Forty-eight primers were designed using PRIMER3 (Rozen and
Skaletsky 2000) and polymorphism was assessed using 32 DNA samples extracted from 4 bloods, 4
muscles, 20 feathers, 3 egg membranes, and 1 pellet collected from wild or zoo birds.
Forward primers were synthesized with an M13 tag sequence (5’-GTTGTAAAACGACGGCCAGT-3’)
for labeling. PCR was conducted in a final volume of 10 µl, containing 1 µl DNA, 5 µl Multiplex PCR
Master Mix (QIAGEN), 0.2 µM of M13-tailed forward primer, reverse primer and FAM-labeled M13
primer, 0.1 µg of T4 gene 32 Protein (Nippon Gene, Tokyo, JPN). The PCR conditions were: 94°C for
15 min; 45 cycles at 94°C for 30s, 55 °C for 45s, 72°C for 45s; then 8 cycles at 94 °C for 30s, 53 °C for
45s, 72°C for 45s, and a 30 min final extension at 72°C. Amplicon size was measured using the ABI
PRISM 3130xl Genetic Analyzer (Applied Biosystems) and Peak Scanner (Applied Biosystems). The
number of alleles, expected heterozygosities (He), observed heterozygosities (Ho), Hardy-Weinberg
equilibrium (HWE) were calculated using GenAlEx 6.5 (Peakall and Smouse 2006) and Genepop-onthe-Web (Rousset and Raymond 1997).
Twenty loci showed good amplification (Table 1). Deviation from HWE was observed at seven
loci. Six out of 32 samples were collected from zoos rather than the wild population which may
explain some of the reduction in observed heterozygosity. These 20 markers are being applied to
Japanese golden eagle genetic studies for future conservation management.
Acknowledgements
We are grateful to Dr. Inoue and Mr. Akomo-Okoue, Kyoto University, for their invaluable comments
and Ms. Ito, NIES, for her technical assistance. We thank Akita Omoriyama Zoo for providing samples.
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