Molecular Ecology Notes (2005) 5, 830– 833 doi: 10.1111/j.1471-8286.2005.01078.

PRIMER NOTE
Blackwell Publishing, Ltd.

Isolation and characterization of twelve polymorphic

microsatellite markers in bottlenose dolphins (Tursiops
truncatus)
P . E . R O S E L ,* V . F O R G E T T A † and K . D E W A R †
*NOAA National Marine Fisheries Service, Southeast Fisheries Science Center, 646 Cajundome Blvd., Suite 234, Lafayette,
LA 70506 USA, †McGill University and Genome Quebec Innovation Centre and Department of Human Genetics, McGill University,
740 Dr-Penfield Ave., Montreal, Quebec, Canada H3A 1A4

Abstract
We developed polymerase chain reaction primers for 12 dinucleotide microsatellite loci in
the bottlenose dolphin, Tursiops truncatus. Seven markers were obtained after hybridiza-
tion screening, and five following random genome sequencing. Orthologous positions
were computed for nine markers on the bovine genome and for seven on the human
genome. The markers are distributed across chromosomes and found in different types of
DNA regions. All 12 loci are polymorphic for Tursiops. Five loci were also polymorphic
in the related species Stenella frontalis and the more distantly related river dolphin, Inia
geoffrensis, indicating these markers will be informative across the Delphinidae and other
cetacean taxa.
Keywords: Cetacean, kinship, microsatellite, population genetics, Tursiops

Received 1 April 2005; revision accepted 13 May 2005

Microsatellite DNA sequences are powerful resources goal was to produce a suite of loci for population-level
for genetic studies of population structure and kinship. studies that would complement those already published
Information provided by these markers can address from T. truncatus (Shinohara et al. 1997; Rooney et al. 1999;
biological questions ranging from the fine scale Caldwell et al. 2002) and to include some that had a rela-
determination of individual identity and relatedness to tively high number of alleles (n > 10) and could be useful in
much broader scale questions, including the determination studies of kinship and relatedness.
of population genetic structure and evolutionary Following the protocols of Estoup & Turgeon (1996),
relationships. However, not all markers are equally genomic DNA isolated from a coastal Atlantic bottlenose
informative at each level of genetic divergence. For studies dolphin (T. truncatus) was digested with Sau3A1, run on a
of relatedness, kinship and assignment, loci with larger 1% low melting point agarose gel and fragments between
numbers of alleles are more informative and provide more 300 and 900 bp were excised from the gel and purified. The
accurate assessments. However, this same feature can be restricted DNA was ligated into pUC18 BAM/BAP (Amer-
problematic in studies of population structure, as loci with sham) and transformed into XL1-Blue cells (Stratagene) via
large numbers of alleles can confound standard analyses of electroporation. Colonies with inserts were individually
population subdivision (O’Reilly et al. 2004; Olsen et al. transferred to grid plates, colony lifts were performed and
2004) and will require large sample sizes to accurately the resulting membranes hybridized with a digoxigenin-
assess population allele frequencies. labelled (CA)10 probe (Roche). Positive colonies were
To aid in studies of genetic structure and relatedness in sequenced directly using universal vector primers. In
small cetacean species, we have developed a suite of 12 all, 3080 colonies were screened, 48 were positive for a CA
microsatellite markers cloned from Tursiops truncatus. Our repeat insert and were sequenced, and primers were
designed for 13 of the sequenced inserts.
Correspondence: P. E. Rosel, Fax: + 1 337 291 2106; In a parallel experiment, we used a HydroShear device
E-mail: patricia.rosel@noaa.gov (Genomic Solutions) to mechanically fragment genomic

© 2005 Blackwell Publishing Ltd

© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 5, 830–833

Table 1 Primer sequences, repeat motif, annealing temperature (Ta) and size range for microsatellite loci cloned from coastal Atlantic bottlenose dolphins, Tursiops truncatus and variability
seen across species. Number of alleles (A) observed across all populations examined within a species; observed (HO) and expected (HE) heterozygosities estimated from single populations
except in offshore T. truncatus where structure is unknown. n, number of individuals used in calculation of HO and HE. GenBank Accession nos DQ18980 – DQ18991. Blank cells denote
species –locus combinations not tested

Size T. truncatus (coastal) T. truncatus (offshore) Stenella frontalis Inia geoffrensis

Repeat Ta range
Locus Primer sequence motif (°C)* (bp) A HO HE n A HO HE n A HO HE n A HO HE n

Ttr04 5′-6-FAM-CTGACCAGGCACTTTCCAC-3′ (CA)25 62 99–123 9 0.674 0.64 92 14 0.806 0.778 248 15 0.658 0.683 73 1 — — 7
5′-GTTTGTTTCCCAGGATTTTAGTGC-3′
Ttr11 5′-TET-CTTTCAACCTGGCCTTTCTG-3′ (CA)21 62 193–223 8 0.713 0.647 94 12 0.802 0.837 248 10 0.569 0.581 72 8 0.702 0.713 205
5′-GTTTGGCCACTACAAGGGAGTGAA-3′
Ttr19 5′-6-FAM-TGGGTGGACCTCATCAAATC-3′ (CA)17 60 171–213 8 0.638 0.688 94 11 0.819 0.867 248 13 0.708 0.793 72 4 0.22 0.340† 50
5′-GTTTAAGGGCTGTAAGAGG-3′
Ttr34 5′-TET-GCACATGAGTATGTGGACAGG-3′ (CA)19 58 183–205 6 0.421 0.446 95 10 0.607 0.688 247 8 0.341 0.426 73 4 0.739 0.64 23
5′-GTTTCCTCCTTGGGAGTGTCCTCT-3′
Ttr48 5′-TET-AAGAGGATGCAAATGGCAAG-3′ (CA)18 58 128–142 7 0.484 0.514 95 8 0.806 0.843 248 10 0.452 0.464 73 4 0.45 0.386‡ 109
5′-GTTTGGTAAGAAAATACCAAAGTCC-3′
Ttr58 5′-HEX-TGGGTCTTGAGGGGTCTG-3′ (CA)17 60 179–197 6 0.547 0.633 96 11 0.789 0.817 246
5′-GTTTGCTGAGGCTCCTTGTTGG-3′
Ttr63 5′-6-FAM-CAGCTTACAGCCAAATGAGAG-3′ (CA)34 60 83–151 16 0.833 0.892 95 25 0.870 0.901 246 7 0.629 0.662 205
5′-GTTTCTCCATGGCTGAGTCATCA-3′
TtrFF6 5′-HEX-AAGTAAGTGCTCCTTTGACTGG-3′ (CA)20 54 155–159 7 0.651 0.692 39
5′-GTTTGGCAGAGAGATATTAGGACAGC-3′
TtrRC12 5′-HEX-GAAAAATGCTTCATGCAAC-3′ (TA)19 58 125–141 9 0.910 0.855 34
5′-GTTTCATGATGGCAAAATGATAC-3′
TtrRH1 5′-HEX-AAGGGGTCTGGAGCAAATGT-3′ (TA)21 50 196–212 8 1.0 0.864 10
5′-GTTTCCACACCTTCTTTGGGGTAA-3′
TtrRC11 5′-TET-CACTTATACCTCTGGAATC-3′ (CT)16 58 145–151 4 0.800 0.679 10
5′-ACATAGACTGGATTTGTCC-3′
TtrRA6 5′-TET-AGTTATCCCCCAGTCACATT-3′ (TG)20 60 126–128 2 0.250 0.533 8

P R I M E R N O T E 831
5′-CTAAGTGAAGGAGCAAGCAA-3′

*Cycling profiles followed 94 °C for 30 s and then 30 cycles at 94 °C 30 s, Ta 30 s, 72 °C 30 s and a 10-min final extension at 72 °C.
†Out of HWE (heterozygote deficiency); ‡Out of HWE (heterozygote excess).
832 P R I M E R N O T E

DNA to a 2–4 kb size range. The DNA was end repaired Table 2 Candidate orthologous positions on the bovine and
with T4 DNA polymerase and ligated into pUC19 vector human genomes for nine of 12 microsatellite loci cloned from
after digestion with SmaI. In all, 1824 random colonies Tursiops truncatus
were picked and sequenced using universal primers in the
Locus Bovine* Human† Closest gene (human)
vector. We identified 32 instances of simple repetitive
sequences of at least 30 bp in length, and flanked by at least Ttr04 211070 chr17 : 29.6 Mb CCL2
50 bp of nonrepetitive sequence on both sides (to permit Ttr11 155471 chr12 : 88.0 Mb DUSP6
primer design). Six of these contained pure dinucleotide Ttr19 238099
repeats. Ttr34 65638 chr1 : 196.4 Mb
Primers for candidate loci were synthesized with the 5′ Ttr48 70161 chr1 : 44.7 Mb FLJ10597
Ttr58 99483 chr12 : 111.7 Mb RPH3A
end of the reverse primer ‘pig-tailed’ (Smith et al. 1995) and
TtrFF6 220067 chr9 : 12.7 Mb TYRP1
the 5′ end of each forward primer synthesized with a fluo- TtrRH1 314377
rescent dye attached. These new microsatellite loci were TtrRC11 90633 chr4 : 79.5 Mb
screened to determine whether they (i) would amplify,
(ii) were polymorphic and (iii) were unlinked and were in *Bovine scaffold from Bos taurus 6 October 2004 genome assembly.
Hardy–Weinberg equilibrium (HWE) as calculated using †Human chromosome and Mb position from Human Genome
genpop 3.4 (Raymond & Rousset 1995). Standard poly- May 2004 (hg17) assembly (http://genome.ucsc.edu).
merase chain reaction (PCR) conditions included 20 mm Tris-
HCl pH 8.4, 50 mm KCl, 1.5 mm MgCl2, 150 mm dNTPs, sampling of the dolphin genome (Table 2). We also
15 pm of each primer, 0.6 U Taq polymerase (Invitrogen) observed that two markers (Ttr48 and Ttr04) were orthol-
and 25–50 ng DNA. Allele sizes were determined using an ogous to similar types of repeats (CA repeats) in the bovine
ABI 310 Genetic Analyser and internal lane standards genome, further indicating that these markers may have
(GS-500 TAMRA) and the genescan software version 3.1 wide applicability across the Cetacea and perhaps even the
(Applied Biosystems). Of the 18 loci for which primers Cetartiodactyla.
were synthesized, 12 passed all three criteria (Table 1).
We tested all 12 loci in T. truncatus, and a subset of the
loci in the closely related Atlantic spotted dolphin Stenella Acknowledgements
frontalis and the more distantly related taxon Inia geoffrensis We gratefully acknowledge the laboratory assistance of L. Adams,
(Table 1). The number of alleles found varied across loci S. Kingston and A. Sellas (NMFS SEFSC Marine Mammal Molecular
and across species. In I. geoffrensis, two loci (Ttr11 and Genetics Laboratory) in optimizing and screening loci, and WICGR
Ttr19) were not in HWE and one (Ttr04) was monomorphic. and MUGQIC sequencing centres for support. We thank V. da Silva
The screening of loci yielded more alleles in S. frontalis and T. Martin for Inia samples, and A. Hohn and the NMFS Strand-
ing group in Beaufort, NC, S. Barco, L. Hansen, W. McFee,
(Adams & Rosel in press) and the offshore morphotype of
K. Mullin and E. Zolman for help with obtaining Tursiops and
Tursiops than in the coastal morphotype. This is likely due
Stenella samples.
to the population sizes of S. frontalis and offshore bottle-
nose dolphins, which are thought to be larger than that of
the coastal bottlenose dolphins used here (Waring et al. References
2004). Our results indicate that these 12 markers will be Adams L, Rosel PE (in press) Population differentiation of the
useful across a wide taxonomic breadth of small cetaceans Atlantic spotted dolphin (Stenella frontalis) in the western North
and particularly within the family Delphinidae. Several Atlantic, including the Gulf of Mexico. Marine Biology.
markers show high levels of polymorphism and will be Caldwell M, Gaines MS, Hughes CR (2002) Eight polymorphic
useful in studies of kinship and relatedness. microsatellite loci for bottlenose dolphin and other cetacean
species. Molecular Ecology Notes, 2, 393–395.
Using the recently compiled assembly of the bovine
Estoup A, Turgeon J (1996) Microsatellite markers: isolation
genome (Bos taurus assembly of 6 October 2004: http://
with nonradioactive probes and amplification (http://
www.hgsc.bcm.tmc.edu/projects/bovine/) and the essen- www.inapg.inra.fr/dsa/microsat/microsat.htm).
tially completed sequence of the human genome (Inter- International Human Genome Sequencing Consortium (2004) Fin-
national Human Genome Sequencing Consortium 2004), ishing the euchromatic sequence of the human genome. Nature,
we used blat (Kent 2002) to determine candidate orthologous 431, 931–945.
locations for nine of the markers on the bovine genome and Kent WJ (2002) blat — the blast-like alignment tool. Genome
Research, 12, 656–664.
seven of the markers on the human genome. Using the
O’Reilly PT, Canino MF, Bailey KM, Bentzen P (2004) Inverse
human positions, we observed that the markers were
relationship between FST and microsatellite polymorphism
distributed across chromosomes and arose from different in the marine fish, walleye pollock (Theragra chalcogramma):
types of genomic regions (intergenic spaces, introns, implications for resolving weak population structure. Molecular
exons), indicating that our techniques provided a random Ecology, 13, 1799–1814.

© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 5, 830– 833

 P R I M E R N O T E 833

Olsen JB, Habicht C, Reynolds J, Seeb JE (2004) Moderately and Shinohara M, Domingo-Roura X, Takenake O (1997) Microsatellites
highly polymorphic microsatellites provide discordant estimates in the bottlenose dolphin Tursiops truncatus. Molecular Ecology,
of population divergence in sockeye salmon, Oncorhynchus 6, 695–696.
nerka. Environmental Biology of Fishes, 69, 261– 273. Smith JR, Carpten JD, Brownstein MJ et al. (1995) Approach to
Raymond M, Rousset F (1995) genepop (version 1.2): population genotyping errors caused by nontemplated nucleotide
genetics software for exact tests and ecumenicism. Journal of addition by Taq DNA polymerase. Genome Research, 5, 312–
Heredity, 96, 248–250. 317.
Rooney AP, Merritt DB, Derr JN (1999) Microsatellite diversity in Waring GT, Pace RM, Quintal JM et al. (2004) U.S. Atlantic and Gulf
captive bottlenose dolphins (Tursiops truncatus). Journal of of Mexico Marine Mammal Stock Assessments — 2003. NOAA Tech-
Heredity, 90, 228–231. nical Memorandum NMFS-NE-182, Woods Hole, Massachnsetts.

© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 5, 830–833