TECHNICAL NOTE

Nine original microsatellite loci in prickly sculpin (Cottus asper)and their applicability to other closely related Cottus species

Jason Baumsteiger • Andres Aguilar

Received: 12 September 2012 / Accepted: 28 September 2012 / Published online: 9 October 2012

� Springer Science+Business Media Dordrecht 2012

Abstract Prickly sculpin (Cottus asper) are a widespread

but largely understudied native freshwater fish in coastal

and inland rivers of Western North America. Given the

extreme anthropogenic changes in this region, prickly

sculpin represent a model organism to study historical and

contemporary changes. We present nine novel microsatel-

lites and four additional loci developed on a distantly

related Cottus species. Loci range in allelic size from one

to eleven and expected heterozygosity from 0.08 to 0.65

within a single inland population. Most loci were geno-

typed on three different prickly sculpin populations and

three closely related sympatric Cottus species allowing for

future comparative studies between and within species.

Keywords Conservation � Population genetics �Pyrosequencing

Contemporary rivers along the westernmost edge of North

America are under intensive anthropogenic influence,

needing improved information to conserve native fresh-

water species (Frissell 1993). Thanks to a wide distribution,

prickly sculpin (Cottus asper) may shed light on past and

present connectivity amongst watersheds (Page and Burr

1991). Early studies suggest the species is divided into

coastal and inland forms (Krejsa 1965), with a suspected

intergrade form (Moyle 2002). We present data from nine

novel microsatellite loci and four previously developed loci

on another species to sufficiently discern population level

differences for this species. Loci are also relatively effec-

tive in amplifying three closely related species of Cottus,

an important factor given the sympatric distribution and

potential hybridization of these species with C. asper

(Moyle 2002).

Three populations of 25–32 individuals of C. asper were

collected from ‘‘inland’’ Kings River (Kings County, CA),

‘‘coastal’’ Redwood Creek (Humboldt Co., CA), and

‘‘intergrade’’ Suisun Bay (Contra Costa Co., CA) popula-

tions. Three additional species were cross-amplified: 25

riffle sculpin (C. gulosus) from the Kings River (Kings Co.,

CA), 30 Pit sculpin (C. pitensis) from the Pit River (Modoc

Co., CA), and six coastrange sculpin (C. aleuticus) from

the Smith River (Del Norte Co., CA). Tissue samples were

taken from lower caudal fin clips and stored in 100 %

ethanol. All genomic DNA was extracted using the DNeasy

Tissue Kit (Qiagen).

Microsatellite markers were developed via two methods:

cloning and pyrosequencing. Cloning followed the enrich-

ment protocol of Glenn and Schable (2005). Nuclear DNA

from eight prickly sculpin was digested with restriction

enzymes and ligated to SuperSNX24 linkers. Mixes 2–4

(from Glenn and Schable 2005) of biotinylated probes were

hybridized to the DNA and captured with Dynabeads (Dynal,

Oslo, Norway). Beads were washed with warmed solutions

of 29 SSC, 0.1 %SDS (29) and 19 SSC, 0.1 %SDS (49).

DNA fragments were released from beads and precipitated

through a combination of high temp (95 �C), 95 % ethanol,

and NaOAc. Resultant DNA was amplified with a poly-

merase chain reaction (PCR) on an Applied Biosystems

(ABI) 2720 Thermal Cycler. Fragments were then cloned

into plasmids using the TOPO TA Cloning Kit� containing

pCR 2.1-TOPO with TOP 10 cells (Invitrogen) followed by

PCRs of bacterial colonies containing vectors and inserts.

Final fragments were cleaned with ExoSAP (USB) and

J. Baumsteiger (&) � A. Aguilar

School of Natural Sciences and Sierra Nevada Research

Institute, University of California Merced, 5200 N. Lake Rd.,

Merced, CA 95344, USA

e-mail: jbaumsteiger@ucmerced.edu

123

Conservation Genet Resour (2013) 5:279–282

DOI 10.1007/s12686-012-9787-2

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280 Conservation Genet Resour (2013) 5:279–282

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sequenced using a BigDye� Terminator v3.1 Cycle

Sequencing Kit (ABI) at the University of California,

Berkeley Core Facility on an ABI 3730 DNA Analyzer.

Pyrosequencing was conducted on genomic DNA

extracted from a single C. asper liver. DNA was shotgun

sequenced on a Genome Sequencer FLX (GS-FLX) System

(Roche Inc.) at the UCLA Genomics Core facility.

Approximately 29,836 reads 100–600 base pairs in length

were obtained and mined for potential Cottus specific

nuclear markers. Raw reads were filtered for adaptor

sequences, length ([400 bp), and subjected to GenBank

BLAST searches to remove sequences with mitochondrial

or bacterial origins. The remaining subset of large sequence

fragments were assembled with CAP3 (Huang and Madan

1999) and prepped for microsatellite searches with MSAT-

COMMANDER (Faircloth 2008).

Cloning provided 10 and pyrosequencing 35 potential

microsatellite sequences. PRIMER3 (Rozen and Skaletsky

2000) identified potential primer pairs flanking di, tri, and

tetra-nucleotide microsatellite sequences. Initial forays

showed four of the cloning and five of the pyrosequencing

primers consistently amplified microsatellites from prickly

sculpin (Table 1). Markers were then tested in different C.

asper populations and across three sympatric species

(Table 2).

Additional Cottus microsatellite primers developed for

C. gobio were also screened in C. asper (Nolte et al. 2005).

While many showed some level of amplification and

polymorphism, four markers showed the most promise

(Tables 1 and 2).

We used the approach of Schuelke (2000) to incorporate

distinct florescent dyes for each locus (Table 1). All

markers were amplified in 10 ll reactions (2 ll of genomic

DNA) with a multiplex kit (Qiagen) or standard PCR mix

of 109 PCR buffer (10 mM Tris–HCl, 50 mM KCl),

1.5 mM MgCl2, 10 mg/ml BSA, 0.25 mM of each dNTP,

and 2U of Amplitaq (ABI). The multiplex kit amplified

primer sets simultaneously according to size and dye (see

Table 1). PCR conditions for the multiplex were 95 �C for

15 min, 20 cycles of 95 �C for 30 s., primer specific

annealing temperature (Table 1) for 30 s and 72 �C for

45 s. followed by 15 cycles with a 48 �C annealing tem-

perature and finally two holds of 60� C for 30 min. and 12�for infinity. The standard PCR varied only by an initial

hold of 5 min and final extension of 72 �C for 7 min.

Fragments were run on an ABI 3,130 9 l genetic analyzer

and scored with ABI GENEMAPPER v3.7 software. Numbers

of alleles and ranges were obtained with CONVERT (Glaubitz

2004). Observed and expected heterozygosity, FIS, Hardy–

Weinberg equilibrium (HWE) and linkage disequilibrium

Table 2 Characterization of microsatellite markers in three forms of C. asper and three closely related sympatric species of Cottus

Species Locus

Ca6 Ca8 Ca65 Ca78 Ca301 Ca316 Ca318 Ca405 Ca414 Cott207 Cott582 Lce219 Lce275

Cottus asper (Inland)

N 25 25 25 25 25 25 25 25 25 25 25 25 25

A 3 3 3 3 5 2 7 3 2 6 3 3 1

Range 190–194 243–255 220–226 155–171 215–243 272–290 249–279 168–180 298–302 309–321 198–206 223–233 307

Cottus asper (Intergrade)

N 26 26 26 15 – 25 2 – – 25 26 25 22

A 4 3 3 2 – 5 2 – – 6 3 11 1

Range 190–198 253–257 220–226 174–182 – 263–278 252–270 – – 309–319 200–204 223–259 307

Cottus asper (Coastal)

N 32 32 32 31 – 32 28 – – 32 32 25 13

A 2 4 1 2 – 1 9 – – 7 2 9 1

Range 190–196 245–257 222 174–182 – 272 246–273 – – 309–323 200–202 229–259 307

Cottus gulosus

N 25 25 25 25 25 25 25 25 25 – 25 25 25

A 2 1 3 1 1 2 2 2 1 – 1 2 1

Range 192–194 243 220–226 155 215 287–290 246–249 168–180 302 – 206 220–230 307

Cottus pitensis

N 30 30 30 30 – 30 22 – – – 30 – –

A 1 1 4 1 – 2 2 – – – 6 – –

Range 192 243 220–228 166 – 248–251 249–270 – – – 210–224 – –

Cottus aleuticus

N 6 6 5 6 – 5 – – – 6 6 6 6

A 7 4 3 4 – 2 – – – 9 4 5 4

Range 184–212 243–255 220–226 158–206 – 260–272 – – – 319–383 198–220 229–253 312–324

Conservation Genet Resour (2013) 5:279–282 281

123

values were obtained with GDA v1.1 (Lewis and Zaykin

2001). Only one locus did not meet HWE expectations

(Cas6) and no linkage disequilibrium was detected after

correcting for multiple tests with a sequential Bonferroni

(Rice 1989).

Overall, markers appear to be robust in C. asper,

showing minor differences between populations (Table 2).

Markers indicate some allelic richness and range differ-

ences in other species, an important factor in differentiating

sympatric species. Additional populations should add to the

range and number of alleles in each species, making these

novel markers a valuable resource in the understanding and

conservation of this poorly studied species.

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