Development of nineteen polymorphic microsatellite loci in the threatened polar bear (Ursus
maritimus) using next generation sequencing
Supplementary Material, Conservation Genetics Resources
Jessica R. Brandt1*, Peter J. van Coeverden de Groot2, Kai Zhao1, Markus G. Dyck3, Peter T.
Boag2 and Alfred L. Roca1
1
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801
USA
2
Department of Biology, Queen’s University, Kingston, ON, K7L 3N6 Canada
3
Department of Environment, Government of Nunavut, Igloolik, NU, X0A 0L0 Canada
*Corresponding Author: jrbrandt@illinois.edu
1
Supplementary methods
Next-generation sequencing (NGS) platforms (e.g. Roche 454 FLX Titanium and
Illumina Genome Analyzer) are being used increasingly for large-scale identification of genetic
markers in species of interest (Csencsics et al. 2010; Saarinen & Austin 2010). NGS methods
provide rapid and effective means for identification of hundreds to thousands of candidate
polymorphic microsatellite loci in any species for which samples are available (Castoe et al.
2010; Lepais & Bacles 2011). This method of characterizing microsatellite markers allows for
the rapid screening of large sequence databases for identification of loci that meet stringent
parameters.
Microsatellite markers designed to amplify very short target regions (< 200 bp) can be
used on samples that are highly degraded or contain low quality DNA, typical of fecal samples,
museum specimens, or confiscated materials (Ishida et al. 2012). These markers can be useful for
obtaining data from non-invasively acquired samples, minimizing the stress and danger of
handling live individuals during specimen collection, while increasing the proportion of a
population from which data can be obtained.
Studies of polar bear population structure and genetic diversity have utilized
microsatellite markers designed in the black bear (Ursus americana), brown bear (Ursus arctos),
Asiatic black bear (Ursus thibetanus), or a combination of these (Paetkau et al. 1995; Paetkau et
al. 1997; Paetkau & Strobeck 1998; Paetkau et al. 1999; Cronin et al. 2006; Crompton et al.
2008; Cronin et al. 2009; Zeyl et al. 2009). While a recent study developed ten microsatellite loci
in the polar bear (Poissant & Davis 2011), only two of the novel loci yielded amplicon sizes that
were less than 200 base pair in length. We therefore sought to generate microsatellite loci with
2
very short amplicons sizes, which would be useful for studies relying on non-invasive
genotyping of polar bears.
One high quality polar bear DNA sample was subject to shotgun sequencing using the
Roche 454 GS FLX+ Titanium kit; a ¼ plate run was completed. The MSATCOMMANDER
program (Faircloth 2008), used for identification of microsatellite repeats and initial primer
design, was used to require a flanking region with a minimum length of 18 bp between the
microsatellite array and the primer sequences. Primers were designed in MSATCOMMANDER
through an interface with PRIMER3 software (Rozen & Skaletsky 2000) to meet the following
criteria: amplification of a target product in the 75 to 150 bp size range (inclusive of the two
primer lengths), optimal length of 20 base pair (range 18 to 22 base pair), optimal melting
temperature of 60.0 °C (range of 58.0 °C to 62.0 °C), optimal GC content of 50%, inclusion of at
least 1 bp GC clamp, low self or pair complementarity and a maximum end stability of 8.0
(Faircloth 2008). Once designed a number of quality checks were implemented before selection
of primer pairs for testing.
To prevent amplification of multiple non-target loci two steps were taken to ensure the
uniqueness of the primer sequences: 1) a Perl script was written to search each primer sequence
against the entire 454 generated sequence database and 2) primer sequences were searched
against the non-redundant BLAST database. Any primers showing evidence of being part of a
repetitive element (e.g. LINEs or SINEs) were removed from further analysis.
DNA extracts from 10 adult male and 10 adult female individuals surveyed as part of the
government of Nunavut’s census of the polar bears of M’Clintock Channel, Nunavut, were used
to assay polymorphism at the novel loci. PCR products were fluorescently labeled using M13tailed primers (CACGACGTTGTAAAACGAC). Primer pairs were initially tested by PCR
3
performed in a 10.76 uL reaction mixture that included: 0.2 uM of each forward and reverse
primer (M13-tailed forward primers), 0.2 mM of each dNTP, 1x PCR buffer, 1.5 mM MgCL2,
0.5 unit of Taq polymerase, 0.5 uL of a primer tailed with a 700 Infrared dye, and 1.2 uL of
template DNA. Two PCR algorithms were used: a) initial 94°C for 5 min; with 35 cycles of 20
sec at 94°C, 30 sec at 55°C, 30 sec at 72°C; with a final extension of 72°C for 10 min or b) initial
94°C for 5 min; with 16 cycles of 20 sec at 94°C, 30 sec of 50°C up to 58°C (0.5°C increase per
cycle), 40 sec at 72°C; followed by 24 additional cycles with 58°C annealing and a final
extension at 72°C for 7 min (detailed PCR mix and algorithms are listed below). PCR amplicons
were separated on a 6.5% polyacrylamide gel using the Licor 4200 automatic sequencer and
allele sizes were scored using Gene ImagIR v.4.05 (Scanalytics).
At total of 81 primer sets were examined for variability. Fifty-nine of the loci assessed
contained dinucleotide repeats, of which 19 successfully amplified a product in the expected size
range and 6 were polymorphic. For an additional 22 loci with non-dinucleotide repeats 14
successfully amplified a product in the expected size range and 13 were polymorphic. Of the 33
primer pairs that consistently amplified products in the correct size range 19 were polymorphic.
Microsatellite variability was assessed based on the number of alleles per locus and expected and
observed heterozygosity calculated respectively by GENEPOP, v.4.0 (Raymond & Rousset
1995) and POPGENE, v. 1.32 (Yeh & Boyle 1997). Linkage disequilibrium between pairs of loci
was calculated with FSTAT, v. 2.9.3.2 (Goudet 1995) and deviations from Hardy-Weinberg
equilibrium were estimated in GENEPOP; statistical significance was evaluated after a
Bonferroni correction (Rice 1989).
4
Details of the PCR setup and PCR algorithm
PCR Components
Volume (ul)
Distilled water
7.90
10X PCR Buffera
1.00
b
dNTP Mix (100mM)
0.02
Forward Primer with M13 Tail (100uM)
0.02
Reverse Primer (100uM)
0.02
a
Taq Polymerase
0.10
700 IR dye tailed primer
0.50
Template DNA
1.20
Total Volume
10.76
Ultrapure Taq DNA Polymerase with 10X ViBuffer S (Vivantis, PL1202)
b
25mM of each dNTP (dATP, dCTP, dGTP, and dTTP) in a mix (Vivantis, NP2406)
a
PCR Algorithms
A. 55°C Anneal
5 min at 94°C
35 cycles of 20 sec at 94°C, 30 sec at 55°C, 30 sec at 72°C
10 min final extension at 72°C
Hold at 4°C
B. Touchup
5 min at 94°C
1 cycle of 20 sec at 94°C, 30 sec at 50.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 50.5°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 51.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 51.5°C, 40 sec at 72°C
5
1 cycle of 20 sec at 94°C, 30 sec at 52.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 52.5°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 53.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 53.5°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 54.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 54.5°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 55.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 55.5°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 56.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 56.5°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 57.0°C, 40 sec at 72°C
1 cycle of 20 sec at 94°C, 30 sec at 57.5°C, 40 sec at 72°C
24 cycles of 20 sec at 94°C, 30 sec at 58.0°C, 40 sec at 72°C
7 min final extension at 72°C
Hold at 4°C
6
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9
B.
Number of loci
Number of loci meeting primer design criteria
A.
Microsatellite Motifs
Microsatellite Motifs
Supplementary Figure S1. Number of identified microsatellite loci for each motif
(panel “A”) and the number of loci of each motif which met the criteria for potential
primer design, i.e., target sites for primers could be found near the repeat motif on
both flanks (panel “B”). Note that the Y axis scales are different for the two panels.
The dinucleotide motif AC was identified most frequently in the sequencing data;
other dinucleotide motifs (AG and AT) were also commonly found. The most
abundant motifs contained the largest overall proportion of loci that had suitable
priming regions that were not part of a repetitive element.
10
Supplementary Table S1. Number and type of microsatellite motifs identified by next
generation sequencing of polar bear DNA.
Repeat Motif
Dinucleotide
Trinucleotide
Tetranucleotide
Pentanucleotide
Hexanucleotide
No.
Repeats
˂10
10
˃10
˂10
10
˃10
˂10
10
˃10
˂10
10
˃10
˂10
10
˃10
Sequenced
Loci
1396
459
2368
77
22
108
231
185
260
23
6
13
1
-
Loci with
Primer Sites
82
27
64
7
2
2
17
8
4
3
-
11
Compound or
Interrupted Loci
44
21
48
2
14
14
8
18
2
-
Supplementary Table S2. Full amplicon sequence at polar bear microsatellite loci.
Locus
Uma14
Uma21
Uma35
Uma40
Uma42
Uma65
Uma73
Uma78
Uma84
Uma95
Uma101
Uma102
Uma127
Uma168
Amplicon Sequence (including primer sequences)
GAGTTCCTCTTCATGCTTCGGTTTTTTCCTTTCCTTTCCTTTCCATTTCCAT
TTCCATTCCATTTCATTTCATTTCATTTCATTTCATTTCATTTCATTTCAAAT
TTCCTTTCATTTCCTATTCCCAGGCCATGTGTGTGAAAG
TCCCATCCATGTGTCCATCCATCCATCCACCTGGCCATCCATCCATCTTT
GTGTCCGTCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATCT
TCTTAAGACCTTTTATGTGCCAGGACAGGTGCAGACAG
TCATCAGCGTCACCTACACCACGGCTCTGTACGCGTCCCTGTCTCCGTG
TGTGTGTGTGTGTGTGTGACAGGCCTCGCCCCCCGCCGCCCGTCTCTTG
GGACGCTTCCCAAGCACGTGTCCTGGTTCCTGTGTCCACCTCTC
ACTTACACCATGGGCTCTCCTGGTTCTGAGGCCTCAGACTGAAACTACAT
GTCTGTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCT
ATCTATCATTGGTCCATATTCTGTTTCTCTGGACGCTGAATACA
TACAGAACCCACAGTCCCAGACGAAGAAACGCTATCTCTTAGCGGATAG
TGGCTCATTCGTTCGTTCGTTCGTTCATTCATTCATTCATTCATTCATTCAT
TCATTCAGTGTTTACTGAGTCCTACTTGAGGCCAATCGTTCTGTTCTG
ACGAAATGTGTTACCCTGCAGTAGGGTCTGCCGTGCTTGTGAGTGAGTG
AGTGAGTGAGTGAGTGAGTGAGTGCTGGCAAAGAGTGCGCTCACCCTTG
ACCACTTGTGGCTGCTCTGACT
CTAGGTGGTCTCCCTCTGTGGCCCTGCATCCATTTTTCTTCCTATATGAAT
CATTAGCATTCATTCATTCATTCATTCATTCATTCATTCAACGAATATCCAC
TGAGCAGCTACTCTGTGCCAGGCACATAGAACAGCT
GAAGAGCAGTCAAAGCCAGGGCAGAGCACGTGGGGTGGGGTGGGAAG
GGAAGGGAAGGGAAGGGAAGGGAAGGGAAGGGAAGGGAAGGGACACG
TGGGGAAAAGAGCTTGGTGCAATCAGAGAACCGAAAGAAGGCC
AGGAGGGCTTCTGAACTGTGGACCTCCCAATCATTCTGAGGTTTTTTTGT
TTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGGGGTTTTTAGGTCTGGAAGC
CAAGTTGGACAACACTTAACAAGAACAATAGCTTCCTTGTTCTCGGG
AGTACAGATCCCGGCACAAGGATGAGCTCAGTGAAGGCTGGTTGCTGAA
ATGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGATGGATG
GATGAGTGGATGGATGGATGGGGATGCCAAAGAATTCTGGCAGGGA
TCCCAGACAAGAAAGCACAGAATTCTGTCGTACAGGAAAAAAAAAGAGA
GAGAGAGAGAGAGAGACTTTTATAAATCACAGCTGTAGGAGAGTGTCTG
CACATAGTAAAATTAGAAGCAGTGGGACATGGAAC
TGAAATCAAGAGCCCGACACTTAACCAACTGCCCCAGTTTCCCTTTCTTT
CTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCT
TCTTTTTTGAGTTTTAGAATGTCCAATCCTTACAAGCACGT
CTGCTTTGCTGGTGGACTTGGTGCCGCTGCTGCTGCTACTGGTGCTGCT
GTTGGGCGGGGGGGCTCGTGGTGGTGGTGGTGGTGGTGGTGGTGGTG
GTGCTGCCGCTGCTGCTGCTGCTGATGCTGGGTGGAGGG
GCCAGGCCTTTGAATTCTGGATGAATTCATTCAAGTACCCGGTATGATAC
CAATATCGTATCTGTCTCCCCAACACACACACACACACACACACACACTT
ATACACACACAATTGTGGCATGACACATGGATGCAGCACCAAC
12
Uma185 ACGTGTCCTAAGGTATGCTGGGAGGCAGGTGTGTGCGTGCTTGTGTGTG
TGTGTGTGTGTGTGTGTGTGTGTGCACACTCATGTATGCACATAGCTTGC
CATGACCAGAGAAC
Uma211 CTCCCTTCTTCCTCTGCCTGCCGCTCCTCTCCTTATGTGTGCTCTCTCTC
TCTCTCTCTCTGTCAAATAAATGAATAAAATCTTTAAGGTGATTTCCCTGT
CTGCATG
Uma218 AGGCCAAGGGTACTACATGCCCTGGACCTGCCTTAGTTCTGCAGGCCGT
GTGTGTGTGTGTGTGTGTGCGTGTAGAACAGACTGTCCTACCACCAGTG
TATGGTGTGGAAAGGAACAGGGGAAAGGGAAGGGAGACGGTGCTCTTA
Uma229 GTCTGGAGCAACACAGGATGTCAGAGGCTCCATTTTGATAGATAGATAGA
TAGATAGATAGATAGATAGATAGATAGATATTTTCATGATCACCATAGCAG
GGGAGAGGAGATGTGGTGAATCA
Uma277 GTGTTCTGATTTCTCCACCTCCTCACTAGTACTTGTTGCTGTTATTATTATT
ATTATTATTATTATTATTATTATTATTATTATATGGAATGCTTCGTGAATTTA
TGTGTTGTCACGTTTGGGAGGGGCTGTGCTGATCTTCCCTA
13