© Springer Science+Business Media B.V. 200810.1007/s10592-008-9571-8
Twenty-one novel tri- and tetranucleotide microsatellite loci for the Amur tiger (Panthera tigris altaica)
College of Life Sciences, Zhejiang University, Hangzhou, 310058, P.R. China
State Conservation Center for Gene Resources of Endangered Wildlife, Zhejiang University, Hangzhou, 310058, P.R. China
Key Laboratory of Conservation Genetics and Reproductive Biology for Endangered Wild Animals of the Ministry of Education, Zhejiang University, Hangzhou, 310058, P.R. China
Received: 11 March 2008Accepted: 20 March 2008Published online: 26 March 2008
Amur tiger is the largest subspecies of tiger in the world and his conservation has also received much attention. In this study, we isolated and characterized twenty-one tri- and tetranucleotide microsatellite markers from this species. The number of alleles for each locus ranged from two to nine in a group of 60 individuals and the observed and expected heterozygosities were 0.333–0.917 and 0.302–0.822, respectively. The overall discrimination power and exclusion probabilities in parentage and paternity testing for these markers were 1.00, 0.9947 and 0.9999, respectively, indicating high-resolution power of microsatellite markers.
KeywordsAmur tiger Microsatellite Polymorphic Exclusionary power
Amur Tiger (Panthera tigris altaica), the largest living cat in the world, lives in the Russian Far East in the Amur-Ussuri region of Primorski and Khabarovski Krais (States) whilst a few are found in northeast of China and Korea (http://www.savethetigerfund.org). In the last half-century we have lost three subspecies of them. The Bali tiger went extinct in the 1940’s, followed by the Caspian tiger in the 1970’s and the Javan tiger in the 1980’s. It is also likely that we have lost the South China tiger in the wild and only a few individuals of this subspecies remain in captivity (Tilson et al. 2004). There are about 400 Amur tigers in the wild but less than 20 wild individuals exist in China (Wang 1998). Therefore, developing polymorphic microsatellites is essential for genetic examination of the Amur tiger and thus promotes his conservation. Microsatellites have been proved to be a reliable marker for conservation genetics (Zhang and Hewitt 2003;Wan et al. 2004). In this study, we isolated 21 novel microsatellite loci for Panthera tigris altaica.
A total of 60 blood samples were collected from Xiongsen Bear and Tiger Village, one of which was used for microsatellite development. Genomic DNA was extracted using a conventional protocol (Sambrook and Russell 2001). Microsatellite enrichment followed the protocols of Fischer and Bachmann (1998) and Bloor et al. (2001) with some modifications. A 400–1,200 bp Sau3A I-restricted fraction was recovered and ligated to linkers Sau3A I-F (5′-GCG GTA CCC GGG AAG CTT GG 3′) and Sau3A I-R (5′-GAT CCC AAG CTT CCC GGG TAC CGC 3′). The ligated fragments were amplified using Sau3A I-F as forward and reverse primers in a 20 μl reaction system composed of 1 μl of template DNA (30–50 ng/μl), 1 U Taq DNA polymerase (TaKaRa), 2 μl of 10 × PCR buffer (TaKaRa), 1.6 μl of 25 mM MgCl2, 2 μl of 20 mM dNTPs, 1 μl of primer (Sau3A I-F). PCR conditions were as follows: 94°C for 5 min, then 30 cycles of 94°C for 30 s, 60°C for 45 s, 72°C for 90 s, and a final period at 72°C for 10 min. The PCR products were hybridized to biotin-labeled probes by incubation 6–8 h at the optimized temperature and then streptavidin-coated magnetic beads (Roche) were added to capture the target fragment with microsatellite repeats. The eluted fragments were cloned into pMD 18-T vector (TaKaRa) and transformed into JM109 Competent Cells (TaKaRa). Recombinant clones were screened by amplification directly from bacterial colonies, using Sau3A I-F and corresponding probe sequence as forward and reverse primers, respectively. Positive colonies were sequenced on automated ABI 3700 DNA sequencer. Primier version 5.0 was used to design primers for the clones containing microsatellite sequences.
A 5′-M13 tail (5′-CAC GAC GTT GTA AAA CGA C) was added to the forward primer of each primer pair to allow fluorescent labeling during amplification reactions. PCR amplification were performed in a 10 μl volume for all primers, which contains 1 μl of 20 mM dNTPs, 1 μl of 10 × PCR buffer (TaKaRa), 1 μl template of DNA (about 10 ng/μl), 0.8 μl of 25 mM MgCl2, 0.5 U Taq DNA polymerase (TaKaRa), 0.4 μl of each 10 μM primer and 1 μl of 1 μM IRD labeled M13 primer (LI-COR). PCR amplification were conducted as: 94°C for 5 min, 30 cycles of 94°C for 30 s, 40 s at optimized primer-specific annealing temperature (Table 1), 72°C for 45 s, and followed by a final 7 min extension at 72°C. The PCR products were loaded on LI-COR 4200 automated DNA Sequencer.
Twenty-one polymorphic microsatellite loci from the Amur tiger
Primer sequence (5′–3′)
T a (°C)
Product size (bp)
Accession No. (GenBank)
The SAGAGT version 3.2 (LI-COR) was used to perform genotyping. Cervus version 2.0 (Marshall et al. 1998) was adopted to calculate the number of alleles, the observed and expected heterozygosities, mean polymorphic information content (PIC) and probability of exclusion (PE). Deviations from Hardy–Weinberg equilibrium and linkage disequilibrium were analyzed using web-based GENEPOP 4.0 (Raymond and Rousset 1995). The discrimination power (DP) of each microsatellite locus and the cumulative DP (CDP) of a set of microosatellite loci were calculated as described by Kloosterman et al. (1993).
Approximately 4,000 colonies were screened and a total of 200 recombinants that potentially contained microsatellite sequences were obtained. We randomly chose 107 colonies for sequencing and 86 ones contained repeat sequences. Seventy primer pairs were designed, of which 50 dyads succeeded in PCR amplification and yielded specific PCR products. Finally, we got 21 pairs of polymorphic primers (Table 1).
The number of alleles per locus ranged from 2 (locus Pati07) to 9 (locus Pati15) with an average of 4.62, presenting a moderate PIC value of 0.546 (Table 2). The observed and expected heterozygosities of these microsatellites ranged from 0.333 to 0.917 (average = 0.605) and from 0.302 to 0.822 (average = 0.608), respectively, indicating a relatively high level of genetic diversity in the Amur tiger. The loci Pati16 and Pati19 showed significant deviation from Hardy–Weinberg equilibrium (P < 0.001). Furthermore, both of them presented lower observed heterozygosity (H O) than expected (H E) (Table 1), probably suggesting existence of inbreeding (Genlous and Björn 2003). Five of the pairwise comparisons among loci (Pati04–Pati08, Pati02–Pati12, Pati05–Pati13 and Pati16–Pati19) exhibited significant linkage disequilibrium (P < 0.001; Table 2). These loci showed their overall values of DP, PE-1 (for parentage testing) and PE-2 (for paternity testing) were 1.00, 0.9947 and 0.9999, respectively (Table 2), indicating high-resolution power of these microsatellite loci. As a result, this set of polymorphic microsatellite loci would provide a powerful tool for the population genetic studies of the Amur tiger. Furthermore, these markers could serve as potential source of microsatellites for other tiger subspecies in the future.
The values of the number of alleles per locus (N A), the observed and expected heterozygosities (H O and H E), polymorphism information content (PIC), discrimination power (DP) and probability of exclusion (PE-1 and PE-2) for the 21 microsatellite loci in the Amur tiger
We thank Xiongsen Bear and Tiger Village of Guilin in China for providing all samples. This work was supported by a special grant from the state forestry administration of China (No. 2005-4-C04).
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