Isolation and characterization of 113 polymorphic microsatellite loci for the Tibetan frog ( Nanorana parkeri ) using ne
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TECHNICAL NOTE
Isolation and characterization of 113 polymorphic microsatellite loci for the Tibetan frog (Nanorana parkeri) using next generation sequencing Cuimin Wang • Xiaoju Hu • Xiangmo Xie • Pengfei Liu • Juha Merila¨ • Qiong Zhang • Meng-Hua Li
Received: 4 April 2013 / Accepted: 12 April 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract We developed and characterized 113 polymorphic microsatellites in the Tibetan frog (Nanorana parkeri) using 454 GS-FLX next generation sequencing technology. These loci were tested in 46 individuals from two N. parkeri populations from the Tibetan plateau. The average number of alleles per locus was 8.09 (range = 2–20). The average observed heterozygosities (HO) per locus in the two populations were 0.58 (range = 0.04–1) and 0.29 (range = 0–0.87), respectively. These microsatellites will be useful for population and conservation genetics studies of N. parkeri and other closely related species. Keywords plateau
Microsatellite Nanorana parkeri Tibetan
C. Wang X. Hu X. Xie P. Liu Q. Zhang (&) M.-H. Li (&) Key Lab of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China e-mail: [email protected] M.-H. Li e-mail: [email protected] C. Wang X. Hu P. Liu College of Life Sciences, University of the Chinese Academy of Sciences, Beijing 100049, China X. Xie College of Life Science, Yangtze University, Jingzhou 434025, Hubei, China J. Merila¨ Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, P.O. Box 65, Helsinki, Finland
Nanorana parkeri is a frog species endemic to the Tibetan plateau and occurs across a relatively narrow latitudinal (28–31°N) and longitudinal (84–97°E) region (Ma et al. 2009). It occupies an extensive altitudinal range from 2850 to 5100 m above sea level (m.a.s.l.), thereby being the amphibian species with the highest altitudinal distribution in the world and the only species of ranid frog found above 4500 m.a.s.l. (Hu et al. 1987; Zhang et al. 2012). However, the information regarding population genetic structure, mating system and dispersal patterns remain unknown due to the lack of molecular genetic markers. We describe here the development of a total of 113 polymorphic microsatellites in N. parkeri using 454 GS-FLX next generation sequencing technology. The markers will be useful for population genetics and genomic investigations of this species. Two adult individuals of N. parkeri were sampled from Maizhokunggar County (29.7539°N, 91.6682°E; 3800 m.a.s.l.), Tibet, China. The genomic DNA was extracted from behind legs using the standard phenol–chloroform extraction protocol (Sambrook and Russell 2001). The two individuals were sequenced using 454 (Roche) GS-FLX genome sequencer and each was sequenced on 1/8 of a Pico Titer plate. We obtained 150008 reads amounting to a total of ca. 53 Mbp (average read length = 359 bp). Sequences containing di-, tri-, tetra-, penta- and hexanucleotide repeat motifs were identified using the Ta
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