Molecular Ecology Notes (2007) 7, 811–813

doi: 10.1111/j.1471-8286.2007.01712.x

PRIMER NOTE

Blackwell Publishing Ltd

Microsatellite DNA primers for the endangered vermilion darter, Etheostoma chermocki, and cross-species amplification in other darters (Percidae: Etheostoma) J . K H U D A M R O N G S A W A T , L . S . H E A T H , H . E . H E A T H and P . M . H A R R I S Box 870345, Mary Harmon Bryant Hall, The University of Alabama, Tuscaloosa, AL 35487, USA

Abstract The endangered vermilion darter (Etheostoma chermocki) is endemic to the Black Warrior River system in the Mobile Basin in Alabama. Restoration and conservation of this species require an understanding of its population genetic structure, which can be characterized using microsatellite DNA. Nine microsatellite loci were developed; eight loci were polymorphic. Although observed heterozygosity was lower than expected heterozygosity in most polymorphic loci, only one locus showed significant deviation from Hardy–Weinberg equilibrium. These nine markers were tested in an additional 24 species of Etheostoma and appear to have sufficient allelic variation to be useful in studies of population genetic structure. Keywords: cross-species amplification, endangered species, Etheostoma, microsatellite DNA, vermilion darter Received 2 November 2006; revision accepted 8 January 2007

The vermilion darter (Etheostoma chermocki) is endemic to the Black Warrior River system in the Mobile Basin in Alabama (Boschung & Mayden 2004). The distribution of this species is limited to the upper reaches of Turkey Creek in Jefferson County, Alabama. Because this species is found only in one area, and their habitat has been degraded due to anthropogenic activities, the vermilion darter is vulnerable to extinction as demonstrated by population surveys from 1995 to 1997 (unpublished reports in Blanco et al. 1995, 1996, Blanco & Mayden 1997, Stiles & Blanchard 2003). An attempt to restore and conserve this species requires understanding its population genetic structure, which can be assessed using microsatellite DNA. Nine pairs of microsatellite primers were developed for use in estimating the genetic population structure of E. chermocki, and possibly other related Etheostoma species. Tissue samples of E. chermocki were obtained from the pelvic fin of 95% ethanol-preserved specimens collected from Turkey Creek, Jefferson County, Alabama, in 1998. Genomic DNA was extracted using QIAGEN DNeasy kit following the manufacturer’s protocol. DNA yields were screened in ethidium bromide stained agarose gels.

Correspondence: P.M. Harris, Fax: 1-205-348-6460; E-mail: [email protected] © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd

Enrichment of the di- and trinucleotide microsatellite loci was performed by the DNA Laboratory at the Savannah River Ecology Laboratory, Aiken, South Carolina, following the method of Glenn & Schable (2005). The enriched samples were ligated into the pCR2.1 TOPO cloning vector (Invitrogen) and electroporated into Escherichia coli TOP-10 cells. Transformants were selected on LB agar plates containing 100 µg/mL ampicillin and 50 µg/mL kanamycin. Plasmids were purified using the QIAGEN plasmid miniprep kit. A total of 36 colonies were isolated, 24 of which were sequenced. Complete sequences of microsatellite regions were determined using forward and reverse primers (M13) in separate sequencing reactions with double-stranded templates. Allelic variation of loci was assessed by using a 10-µL fluorescence-labelled reaction containing 1 µL of template DNA, 0.8 µL of each primer, 2 µL of BigDye (3.1), 1 µL of BigDye Terminator buffer, and 5.2 µL of distilled water. Sequencing reaction consisted of 40 cycles of 96 °C for 30 s, followed by 50 °C for 30 s, and 60 °C for 4 min. Thirteen pairs of microsatellite primers were developed; nine were optimized for E. chermocki. Two polymerase chain reaction (PCR) protocols were followed based on the methods by Mavárez & González (2006). The first protocol included 12.5-µL reactions containing c. 40–50 ng of DNA template, 5 pmol of the reverse primer, 5 pmol of forward primers consisting of

812 P R I M E R N O T E Table 1 Microsatellite DNA primer sequences in Etheostoma chermocki

Locus Eche001* Eche002* Eche005 Eche008* Eche009*M Eche010* Eche011* Eche012 Eche013

GenBank Accession no.

Primers (5′–3′)

EF1173 12 EF1173 13 EF1173 14 EF1173 15 EF1173 16 EF1173 17 EF1173 18 EF1173 19 EF1173 20

F: M13+TTCGGTGACAGATCAGATTAG R: TCAAACAAAGCAGCAGC F: M13+CCCTTCCTGAGATGGTATAAT R: CCAAAGCTGCAGATACTGATG F: AGCAGAATCACGTTTTCCCAG R: ACCGTCGGGATGGATG F: M13+TGCCTCTTTAGTTTCCC R: CGGTCATATCATCTGCTTTTG F: M13+TCTTTGCTGCTCTTAATTATT R: GACAACAGGACGCTGATG F: M13+CAGTCTTGGCAATTTAACTAA R: CGTCACTCCCTCCCTCT F: M13+CCAAATATGAAACAGCACTT R: TTAGCCTTCACATGGACTATA F: ATTGGCCTAAGGGTTTATGA R: CGTCGGAGTGACTCAGGGTGT F: CGCAGTGTCTCCACATTG R: ACGTCTTCACACATGTCTTCA

Repeat motifs

Size (bp) including Ta (°C) primers†

n

EPICENTRE buffer

(TAC)21

63

145–165

10

C

0.941 0.856

(CAT)14(CTT)5 (CAT)5 (CA)2(GACA)5 CAGA(CA)11 (GGT)3GCG(TG)27

58

185–213

5

C

0.588 0.630

59

115–130

2

C

0.382 0.627

62

161–185

7

D

0.735 0.798

HO

HE

(CT)2CC(CT)4(CA)2CT 57 (CACT)3(CT)3(CA)11 (GA)21 63

213

1

D

—

—

145–165

5

C

0.471 0.725

(CA)18

62

217–225

3

C

0.147 0.671**

(GT)17(GC)28 (AT)2(GT)10 (CA)2(GACA)8(CA)18

55

165–211

19

J

0.853 0.931

62

154–180

7

J

0.647 0.781

*, three primers used in PCR; M, monomorphic; Ta, annealing temperature; †, estimated from 34 individuals; n, number of alleles, HO, observed heterozygosity; HE, expected heterozygosity; ** , significant deviation from HE (P < 0.05); M13 primer, 5′-GTAAAACGACGGCCAG-3′.

4% of M13-tailed forward primer (M13 forward sequence and microsatellite forward primer) and 96% of fluorescently labelled (6-FAM) M13 forward primer, 200 µm each of the deoxynucleotidetriphosphates (dNTPs) and magnesium (EPICENTRE buffers), and 0.25 U of Taq DNA polymerase in commercial buffer (EPICENTRE). The second protocol used two, unmodified primers: 5 pmol of each primer were used, with the forward primer being labelled (6-FAM). PCR was performed in a GeneAmp PCR System 9700 (Applied Biosystems). Thermal cycling conditions consisted of an initial denaturation at 94 °C for 2 min, followed by 25 cycles of 94 °C for 1 min, annealing temperature for 1 min (Table 1), extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min. Primer sequences and optimal annealing temperatures are listed in Table 1. Allele lengths were estimated using an ABI PRISM 310 Genetic Analyser (Applied Biosystems) with genescan-500 TAMRA size standard (Applied Biosystems). All 34 samples of E. chermocki were successfully genotyped. Among nine loci examined, eight loci were polymorphic while one locus (Eche009) was monomorphic. This locus was, however, polymorphic with the optimized PCR conditions in related species (Table 2). Number of alleles at each locus, their estimated size, observed and expected heterozygosities, and deviations from the Hardy– Weinberg equilibrium (HWE) were estimated using arlequin 3.01 (Excoffier et al. 2005). No significant linkage disequilibrium was observed between pairs of studied loci

(P > 0.05). Of eight polymorphic loci, only one locus (Eche011) displayed significant deviations from Hardy– Weinberg equilibrium (P < 0.05), which may be due to the presence of null alleles commonly found in microsatellite loci. Cross-species amplifications for all nine primer pairs were conducted to determine efficacy in an additional 24 species of Etheostoma (Table 2). The results indicated that these primers could be used in population genetic studies of other Etheostoma species with additional optimizations of PCR conditions.

Acknowledgements We are grateful to Dr Wallace Holznagel for sharing his expertise in molecular techniques and Dr Bernard R. Kuhajda for providing specimens of several species of Etheostoma.

References Boschung HT, Mayden RL (2004) Fishes of Alabama. Smithsonian Institute, Washington, D.C. Excoffier Laval LG, Schneider S (2005) arlequin version 3.0: an integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online, 1, 47–50. Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. Methods in Enzymology, 395, 202–222. Mavárez J, González M (2006) A set of microsatellite markers for Heliconius melpomene and closely related species. Molecular Ecology Notes, 6, 20–23. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd

P R I M E R N O T E 813 Table 2 Cross-species amplification with microsatellite primers developed for Etheostoma chermocki using PCR conditions optimized for E. chermocki Locus Eche001 Etheostoma species

N n

SR

Eche002

Eche005

Eche008

Eche009 Eche010

Eche011

Eche012

Eche013

n SR

n SR

n

n

n SR

n SR

n

SR

Subgenus Ulocentra E. baileyi 2 1 148 X * X E. bellator 47 13 181–234 3 197–203 1 130 10 170–210 E. sp. cf. bellator† 7 2 151, 159 2 183, 185 1 130 8 176–260 E. colorosum 2 3 154, 164 * * X E. pyrrhogaster 2 2 133, 155 3 174–185 * X E. rafinesquei 2 2 161, 167 1 132 * X E. ramseyi 4 3 133–154 1 168 1 130 X E. simoterum 2 2 123, 141 1 159 * X E. zonistium 2 1 156 3 173–245 * 3 151–167 Subgenus Catonotus E. kennicotti 1 X * * X Subgenus Doration E. stigmaeum 2 X * 2 130, 146 X Subgenus Fuscatelum E. parvipinne 2 3 166–176 X * X E. phytophilum 2 X X * X Subgenus Litocara E. sagitta 1 2 121, 139 * X X Subgenus Nothonotus E. chlorobranchium 2 X * * X E. douglasi 2 X * 2 115, 130 X E. jordani 1 X * 2 115, 130 X Subgenus Oligocephalus E. caeruleum 2 2 125, 144 2 117, 126 2 130, 144 X E. swaini 2 2 137, 145 1 145 * X E. whipplei 1 1 141 1 144 * X Subgenus Poecilichthys E. euzonum 1 1 139 * X X E. tetrazonum 1 X X * X Subgenus Vaillantia E. chlorosoma 1 1 109 * 2 115, 130 X Subgenus Villora E. edwini 1 * * 2 113, 128 X

SR n

* * * * * * * * *

SR

* 16 144–168 3 152–166 * X X * X *

2 1 2 2 1

* * * X 191, 197 224 191, 199 191, 199 189

3 2 1 2

SR

X * * * 122–154 139, 143 105 107, 113 *

* * * * * * * * *

*

X

X

X

X

*

X

*

X

X

* *

X X

X X

X X

X X

*

*

X

X

X

* * *

2 136, 146 * *

X X X

X X *

X X X

* * *

* 1 167 X

X X X

X X X

X X X

* *

X *

* *

X *

X X

*

X

X

*

*

*

*

X

*

X

†E. sp. cf. bellator (Locust Fork); N, number of individuals; n, number of alleles at each locus; SR, size range of alleles; X, no amplification; *, weak amplification.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd