Y. Sato, H. Ito, R. Ogden, M. Inoue-Murayama

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 020-0857, 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:

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).Amongthe 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-on-the-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.

References

Faircloth BC (2008) Msatcommander: detection of microsatellite repeat arrays and automated, locus-specific primer design. Mol Ecol Resour 8:92–94 doi:10.1111/j.1471-8286.2007.01884.x

Peakall ROD, Smouse PE (2006) GenAlEx 6: genetic analysis in excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295 doi:10.1111/j.1471-8286.2005.01155.x

Rousset F, Raymond M (1997) Statistical analyses of population genetic data: new tools, old concepts. Torends Ecol Evol 12:313–317 doi:10.1016/s0169-5347(97)01104-x

Rozen S, Skaletsky H (2000) Primer3 on WWW for general users and for biologists programmers. Methods Mol Biol 132:365–386 doi:10.1385/1-59259-192-2:365

SRGE (The Society for Research of Golden Eagle) (2014) Population and breeding success of the golden eagle Aquila chrysaetos in Japan, 1981 to 2010. Aquila chrysaetos 25:1–13 (In Japanese, English abstract)