Conservation Genet Resour (2014) 6:59–61 DOI 10.1007/s12686-013-0003-9
MICROSATELLITE LETTERS
Development of nineteen polymorphic microsatellite loci in the threatened polar bear (Ursus maritimus) using next generation sequencing Jessica R. Brandt • Peter J. Van Coeverden de Groot Kai Zhao • Markus G. Dyck • Peter T. Boag • Alfred L. Roca
•
Received: 26 June 2013 / Accepted: 19 July 2013 / Published online: 29 July 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Habitat loss caused by the reduction of Arctic ice threatens polar bear (Ursus maritimus) populations. To aid in polar bear conservation, we used stringent marker design criteria with next generation shotgun sequencing to design primers for 19 novel polymorphic microsatellite loci. The number of alleles per locus ranged from 2 to 7, and the average observed heterozygosity across all loci was HO = 0.67. No linkage disequilibrium was detected between loci and only one locus deviated from Hardy– Weinberg equilibrium. Since genetic studies of wildlife species often rely on non-invasive fecal sampling, the microsatellite markers were designed to amplify short regions (\200 bp) to maximize the potential genotyping success when using low quality DNA. These novel markers will be useful for population and conservation genetic studies of polar bear populations. Keywords Conservation Next generation shotgun pyrosequencing Non-invasive methods
Electronic supplementary material The online version of this article (doi:10.1007/s12686-013-0003-9) contains supplementary material, which is available to authorized users. J. R. Brandt (&) K. Zhao A. L. Roca Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Drive, Urbana, IL 61801, USA e-mail:
[email protected] P. J. Van Coeverden de Groot P. T. Boag Department of Biology, Queen’s University, Kingston, ON K7L 3N6, Canada M. G. Dyck Department of Environment, Government of Nunavut, Igloolik, NU X0A 0L0, Canada
The polar bear (Ursus maritimus) has a circumpolar distribution determined by the formation of sea ice in the Arctic. Included as a threatened species under the Endangered Species Act, the total polar bear population is estimated as 20,000–25,000 individuals. Global climate change is causing a reduction in the sea ice that polar bears rely on for survival, and as a result the population is expected to decline by more than 30 % over the next 45 years (Derocher et al. 2004; Schliebe et al. 2008). This habitat loss is a major threat to polar bears especially given their low reproductive rates and long generation times (Schliebe et al. 2008). Here, we used next-generation sequencing to develop markers for 19 variable microsatellite loci in the polar bear. To facilitate their potential use with non-invasively collected samples, we designed the primers to target short amplicons, which are more suitable for use with DNA of poor quality (Ishida et al. 2012). Total genomic DNA isolated from one polar bear tissue sample was submitted for library preparation and shotgun sequencing on the Roche 454 Genome Sequencer FLX ? platform. A total of 124,557 reads, with an average length of 469 bp, were obtained. Sequence data were screened for di-, tri-, tetra-, penta-, and hexanucleotide microsatellite motifs, each with a minimum of 8 tandem repeats, in MSATCOMMANDER 1.0.8 (Faircloth 2008). Microsatellite motifs with 8 or more repeats were identified in 5330 of the reads, thus 4.3 % of the total reads contained the target repeat regions. Flanking primer pairs were designed with stringent criteria using the PRIMER3 (Rozen and Skaletsky 2000) interface in MSATCOMMANDER, targeting amplicon sizes between 75 and 150 bp (inclusive of the primer lengths). The MSATCOMMANDER program (Faircloth 2008) was modified to require a flanking region with a minimum length of 18 bp between the microsatellite array and the primer sequences. Suitable
123
60
Conservation Genet Resour (2014) 6:59–61
Table 1 Characteristics of 19 polymorphic polar bear microsatellite markers Locus
Repeat motif
Primer sequences
Size (bp)
A
HE
HO
Uma14
(AAATG)8
F: GAGTTCCTCTTCATGCTTCGG
150–170
4
0.63
0.70
150–162
4
0.64
0.90
154–158
3
0.55
0.60
156–168
4
0.71
0.35
160–176
5
0.73
0.80
R: CTTTCACACACATGGCCTGG Uma21
(ATCC)9
Uma35
(AC)10
F: TCCCATCCATGTGTCCATCC R: CTGTCTGCACCTGTCCTGG F: TCATCAGCGTCACCTACACC R: GAGAGGTGGACACAGGAACC
Uma40
(AGAT)12
F: ACTTACACCATGGGCTCTCC R:TGTATTCAGCGTCCAGAGAAAC
Uma42
(AATG)8
Uma65
(ACTC)8
F: ACGAAATGTGTTACCCTGCAG R: AGTCAGAGCAGCCACAAGTG
132–140
3
0.53
0.80
Uma73
(AATG)8
F: CTAGGTGGTCTCCCTCTGTG
156–164
3
0.57
0.60
145–160
4
0.75
0.60
169–177
3
0.62
0.85
154–170
5
0.76
0.90
147–149
2
0.43
0.50
147–175
7
0.77
0.75
150–154
3
0.57
0.81
F: TACAGAACCCACAGTCCCAG R: CAGAACAGAACGATTGGCCTC
R: AGCTGTTCTATGTGCCTGGC Uma78
(AAGGG)9
F: GAAGAGCAGTCAAAGCCAGG R: GGCCTTCTTTCGGTTCTCTG
Uma84
(AAAC)8
F: AGGAGGGCTTCTGAACTGTG R: CCCGAGAACAAGGAAGCTATTG
Uma95
(ATCC)9
Uma101
(AG)10
F: AGTACAGATCCCGGCACAAG R: TCCCTGCCAGAATTCTTTGG F: TCCCAGACAAGAAAGCACAG R: GTTCCATGTCCCACTGCTTC
Uma102
(AAAG)15
F: TGAAATCAAGAGCCCGACAC R: ACGTGCTTGTAAGGATTGGAC
Uma127
(ACC)10
Uma168
(AC)13
F: GCCAGGCCTTTGAATTCTGG R: GTTGGTGCTGCATCCATGTG
160–184
6
0.77
0.80
Uma185
(AC)15
F: ACGTGTCCTAAGGTATGCTGG
131–135
3
0.68
0.57
124–128
2
0.36
0.45
164–168
3
0.61
0.50
138–150
4
0.64
0.70
161–170
4
0.64
0.55
F: CTGCTTTGCTGGTGGACTTG R: CCCTCCACCCAGCATCAG
R: GTTCTCTGGTCATGGCAAGC Uma211
(AG)10
F: CTCCCTTCTTCCTCTGCCTG R: CATGCAGACAGGGAAATCACC
Uma218
(AC)10
F: AGGCCAAGGGTACTACATGC R: TAAGAGCACCGTCTCCCTTC
Uma229
(AGAT)10
Uma277
(AAT)13
F: GTCTGGAGCAACACAGGATG R: TGATTCACCACATCTCCTCTCC F: GTGTTCTGATTTCTCCACCTCC R: TAGGGAAGATCAGCACAGCC
The number of repeat motifs is reported from the 454 data The size indicates the size range of alleles in nucleotide base pairs (includes the primer lengths) A, total number of alleles detected; HE, expected heterozygosity; HO, observed heterozygosity
priming regions were identified for 293 of the candidate microsatellite loci (5.5 % of reads with microsatellite motifs, 0.2 % of total reads). From the identified 293 candidate loci 77 were removed due to the presence of 3 or more identical matches in the original sequencing database (suggesting the loci were in repetitive DNA regions),
123
resulting in a total of 216 loci suitable for testing. Microsatellite motifs varied considerably in both length and nucleotide composition (Supplementary Figure S1; Supplementary Table S1). Nineteen of 81 primer pairs assessed for variability by PCR of DNA extracted from 20 polar bear tissue
Conservation Genet Resour (2014) 6:59–61
specimens were polymorphic (Table 1; Supplementary Table S2). Details regarding experimental procedures are available in the supplementary material. Microsatellite variability was evaluated by number of alleles per locus, expected heterozygosity, and observed heterozygosity. Allelic diversity ranged from 2 to 7 alleles per locus with an average of 3.8 alleles. Average observed heterozygosity across all loci was HO = 0.67, and the average expected heterozygosity was HE = 0.63. Linkage disequilibrium between pairs of loci was calculated and deviations from Hardy–Weinberg equilibrium were estimated. No evidence of deviation from Hardy–Weinberg equilibrium was detected after correction for multiple comparisons with the exception of marker Uma40. No significant linkage disequilibrium was found between pairs of loci. In summary, highly stringent primer design criteria and total amplicon length restrictions resulted in a low percentage of microsatellite loci having suitable priming regions; nonetheless, more than 200 candidate loci were identified. The 19 novel microsatellite loci characterized by this study utilizing high-quality DNA should be useful for examining the structure, dynamics, size estimates, and diversity of polar bear populations. Evaluation of these markers for the amplification of DNA from polar bear fecal samples is ongoing. Very short regions of DNA are targeted, so the markers presented here may be expected to
61
have higher genotyping success with degraded DNA than microsatellite markers with longer target amplicons (Ishida et al. 2012). Thus these markers may also prove useful for conservation genetic studies of polar bear populations using non-invasively collected samples. Acknowledgments Sample collection was funded by the Government of Nunavut and was in full compliance with required permits. This research was funded by the Nunavut General Monitoring Plan and the National Science and Engineering Research Council of Canada. We thank Miranda Hunt for her assistance in optimizing the primers.
References Derocher AE, Lunn NJ, Stirling I (2004) Polar bears in a warming climate. Integr Comp Biol 44(2):163–176 Faircloth BC (2008) MSATCOMMANDER: detection of microsatellite repeat arrays and automated, locus-specific primer design. Mol Ecol Res 8(1):92–94 Ishida Y, Demeke Y, de Groot PJVC, Georgiadis NJ, Leggett KEA, Fox VE, Roca AL (2012) Short amplicon microsatellite markers for low quality elephant DNA. Cons Genet Res 4(2):491–494 Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386 Schliebe S, Wiig O, Derocher AE, Lunn N (2008) Ursus maritimus. IUCN red list of threatened species. http://www.iucnredlist.org/ details/22823/0. Accessed 10 May 2013
123
