Conserv Genet (2009) 10:1121–1123 DOI 10.1007/s10592-008-9723-x
TECHNICAL NOTE
Isolation and characterization of microsatellite loci in the Amami rabbit (Pentalagus furnessi) Junco Nagata Æ Youichi Sonoda Æ Keiko Hamaguchi Æ Naoki Ohnishi Æ Soh Kobayashi Æ Ken Sugimura Æ Fumio Yamada
Received: 7 September 2008 / Accepted: 9 October 2008 / Published online: 1 November 2008 Ó Springer Science+Business Media B.V. 2008
Abstract We report on the isolation and characterization of eight polymorphic and five monomorphic microsatellites in the Amami rabbit (Pentalagus furnessi). Microsatellite polymorphism was determined using 25 individuals. There were 2–11 alleles for each polymorphic locus with heterozygosity ranging between 0.08 and 0.76. Linkage disequilibrium was not suggested between any pairs among the eight polymorphic loci. We suggest that these primers be used in future studies to monitor population size, determine dispersal patterns, and genetic diversity within and between populations of this and related species. Keywords Amami rabbit Pentalagus furnessi Microsatellite Endangered species
The Amami rabbit is considered to be one of the oldest species of Leporidae (Corbet 1983). They are endemic to Amami-Ohshima Island and Tokunoshima Island, in
J. Nagata (&) K. Sugimura Forestry and Forest Products Research Institute, Tsukuba 305-8687, Japan e-mail:
[email protected] Y. Sonoda National Institute for Land and Infrastructure Management, Ministry of Land, Infrastructure, Transport and Tourism, Tsukuba 305-0804, Japan K. Hamaguchi N. Ohnishi F. Yamada Kansai Research Center, Forestry and Forest Products Research Institute, Fushimi, Kyoto 612-0855, Japan S. Kobayashi Environmental Science Research Laboratory, Central Research Institute of Electronic Power Industry, Abiko 270-1194, Japan
southern Japan, and were categorized as endangered by IUCN in 2000. Their populations are declining due to habitat fragmentation and introduced mammalian predators (Sugimura et al. 2000; Yamada et al. 2000; Yamada and Cervantes 2005). Their total numbers have been estimated to be 2,000–4,800 in a survey from 2002 to 2003 (Sugimura and Yamada 2004). Concern about the genetic diversity, genetic structure, and population persistence throughout the species’ range is increasing. The mean litter size of this species is only one (Yamada and Cervantes 2005). Juveniles remain in/around the nest burrow for 3–4 months after parturition and are then driven from the burrow by the mother (Yamada and Cervantes 2005). Little is known about subsequent dispersal. Although microsatellite markers are powerful tools for examining genetic diversity and population genetic structure, markers specific to Amami rabbits are currently not available. We isolated eight polymorphic and five monomorphic microsatellites to facilitate the study of these conservation issues. Microsatellites were isolated following the method of Hamaguchi et al. (2007). Genomic DNA from two rabbits was extracted from ethanol-preserved ear samples collected from Amami-Ohshima Island, Japan, using the QIAamp DNA Mini Kit (QIAGEN). Extracted DNA was digested with Sau3AI, ligated to Linker Sau3AI Cassette (TaKaRa) and PCR amplified over 20 cycles under the following condition: 72°C for 5 min and 94°C for 2 min prior to the first denaturation step for 30 s, annealing temperature at 55°C for 30 s, extension for 4 min, using Cassette Primer C1 (TaKaRa) and LA Taq DNA Polymerase (TaKaRa). The PCR product was hybridized with 30 biotin-labeled oligonucleotide (CA)12 and subjected to magnetic bead capture using super paramagnetic beads (Dynabeads M-280 Streptavidin; Dynal) (Fischer and
123
1122
Conserv Genet (2009) 10:1121–1123
Table 1 Characterization and amplification conditions of eight polymorphic microsatellite loci developed for Pentalagus furnessi Locus
F/R
Primer sequences (50 –30 )a
Repeat motif
PF002
F2
GGAGGGAATCAGCAGATGAG
(AC)2 AA (AC)5 AA (AC)13
R1
TGAATACCACATGGCCTGAA
PF010
F1
TTTCATCATTAAGGCCGACAT
R1
AGGGATACTGAGGGGCAAAT
F1
ATGGCAGAGTCTGGACCACT
R1
AGGTCACGTTCCTTGATGCT
F2 R2
CCAAGGTAGCCCTTCCATTT TTGCCATTTGGGAAGTGAAT
(AG)14G(GA)5
PF015 PF028 PF033
F1
CAGGGCTGTGCCATATACAG
R2
CTTGAAGGGTTGGTGAGGAC
PF070
F1
TCCAGGTTGGCTGGGTATAG
R1
TGCAAAGTCAATGGGGTAGC
PF088
F1
AGGCATTCATGACTGAAATCAA
R1
GACTCATTTCTCACTTGCACTCA
F2
TGGGCAGACTTTGCTCTTCT
R2
GAGCCAGGAGGAGAGAGCTT
PF098
Na
Allele size range (bp)b
HO
HE
GeneBank accession no.
5
214–224
0.6
0.7
AB445478
(AC)13
3
144–150
0.56
0.54
AB445479
(AG)3 GC (TG)17
5
218–230
0.52
0.52
AB445480
11
157–185
0.76
0.82
AB445481
(CA)16
4
158–166
0.44
0.57
AB445482
(AC)13
2
157–159
0.08
0.27
AB445483
(AC)16
5
153–161
0.32
0.38
AB445484
(GT)12(TG)5
4
185–191
0.72
0.67
AB445485
Number of alleles (Na), allele size range, observed heterozygosity (HO), and expected heterozygosity (HE) are given for each locus. Each locus was genotyped using 25 rabbits a
Reverse primers include seven base tail sequence (Applied Biosystems, Japan)
b
Allele size contains seven base pair tail sequence
Bachmann 1998) and amplified by 30 cycles of PCR under the following conditions: denaturation step for 30 s, annealing temperature at 55°C for 30 s, extension for 80 s using Cassette Primer C1 and rTaq DNA polymerase (TaKaRa). PCR products were ligated into a plasmid pGEM-T easy vector (Promega) and transformed in XL1Blue MRF’ supercompetent cells (Stratagene). Inserts were amplified by colony-direct PCR using M13(-20) forward and M13 reverse primers, and 100 clones with inserts were sequenced on an ABI3100 sequencer (Applied Biosystems) using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). Twenty four microsatellite regions were selected for primer design using Primer3-web 0.3.0 (Rozen and Skaletsky 2000). Each microsatellite locus was initially tested for amplification and polymorphism using 11 individuals from Amami-Ohshima Island (n = 10) and Tokunoshima Island (n = 1), Japan. PCR was carried out in 10 ll of reaction mixture, containing 0.5 ll of template DNA, 0.5 lmol of fluorescent-labeled forward primer (Applied Biosystems, Japan), 0.5 lmol of non-labeled tailed reverse primer (Applied Biosystems, Japan), 1.25 U of Ex Taq DNA polymerase, 19 buffer with 2.0 mM MgCl2, and 0.2 mM of each dNTP (TaKaRa). PCR reactions were performed in a GeneAmp 9700 (Applied Biosystems) under the following conditions: 94°C for 2 min; 27 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s; 72°C for 10 min. The amplified products were detected on an ABI 310
123
Genetic Analyzer and fragment size was estimated using GENESCAN 2.1 software (Applied Biosystems). Five loci [PF016 (GeneBank, Accession no AB445486), PF017 (AB445487), PF038 (AB445488), PF072 (AB445489), and PF080 (AB445490)] were found to be monomorphic, while eight loci were found to be polymorphic (Table 1). These eight loci were subsequently genotyped in an additional 14 individuals from Amami-Ohshima Island. The number of alleles for the eight loci ranged from 2 to 11 (average 4.9). Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium were tested using GENEPOP on the web (Raymond and Rousset 1995). Locus PF070 deviated strongly from HWE (P = 0.0043) while all other loci were in HWE (P [ 0.01). Linkage disequilibrium was not suggested between any pairs among the eight loci. Microsatellite makers described here should be useful to monitor population size, and to determine dispersal patterns and genetic diversity within and between populations of this species.
Acknowledgments We thank R. P. Shefferson for laboratory support and for help with editing this manuscript, R. Matsuki for useful suggestions and S. Abe for collecting the samples for this study. This work was supported by the Program for Supporting Activities for Female Researchers funded by the Special Coordination Fund for Promoting Science and Technology of Ministry of Education, Culture, Sports, Science and Technology, Japan. This research was supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B), 17310137, 2005.
Conserv Genet (2009) 10:1121–1123
References Corbet GB (1983) A review of classification in the family Leporidae. Acta Zool Fenn 174:11–15 Fischer D, Bachmann K (1998) Microsatellite enrichment in organisms with large genomes (Allium cepa L.). Biotechniques 24:796–802 Hamaguchi K, Matsumoto T, Maruyama M, Hashimoto Y, Yamane S, Itioka T (2007) Isolation and characterization of eight microsatellite loci in two morphotypes of the Southeast Asian army ant, Aenictus laeviceps. Mol Ecol Notes 7:986–987. doi:10.1111/ j.1471-8286.2007.01746.x Raymond M, Rousset F (1995) GENEPOP VERSION 1.2; population genetics software for exact tests and ecumenicism. J Hered 86:248–249 Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds)
1123 Bioinformatics methods and protocols: methods in molecular biology. Humana, Totowa, pp 365–386 Sugimura K, Yamada F (2004) Estimating population size of the Amami rabbit Pentalagus furnessi based on fecal pellet counts on Amami Island, Japan. Acta Zool Sin 50:519–526 Sugimura K, Sato S, Yamada F, Abe S, Hirakawa H, Handa Y (2000) Distribution and abundance of the Amami rabbit Pentalagus furnessi in the Amami and Tokuno Island, Japan. Oryx 34:198– 206. doi:10.1046/j.1365-3008.2000.00119.x Yamada F, Cervantes A (2005) Pentalagus furnessi. Mamm Species 782:1–5. doi:10.1644/782.1 Yamada F, Sugimura K, Abe S, Handa Y (2000) Present status and conservation of the endangered Amami rabbit Pentalagus furnessi. Tropics 10:87–92
123
