Conserv Genet (2009) 10:1869–1871
DOI 10.1007/s10592-009-9838-8
TECHNICAL NOTE
Isolation and characterisation of 11 microsatellite loci
in the abyssal carapine grenadier Coryphaenoides carapinus
(Actinoperygii, Macrouridae) and cross-amplification
in two other deep-sea macrourid species
Matthias Schneider Æ Hanne Sannæs Æ
Per Erik Jorde Æ Halvor Knutsen
Received: 18 December 2008 / Accepted: 21 January 2009 / Published online: 11 February 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Microsatellites represent an important tool for
characterising population structure, for attributing individuals to stocks, and for revealing ecological processes
taking place on population and meta-population levels. A
sound knowledge of population structure is essential for
sustainable management of exploited fish stocks, and helps
to understand population connectivity and speciation. We
developed for the first time primers for microsatellite loci
in the carapine grenadier, Coryphaenoides carapinus,
inhabiting the abyssal Atlantic. Eleven microsatellites were
obtained from partial genomic DNA libraries enriched for
tetranucleotide repeats. The loci were characterised in three
unrelated individuals and nine loci were found to be
polymorphic. Cross-amplification in two commercially
exploited deep-sea macrourid species (Coryphaenoides
rupestris and Macrourus berglax) resolved two polymorphic loci in each species.
Keywords Coryphaenoides carapinus Coryphaenoides rupestris Macrourus berglax Grenadier Microsatellite primers Polymorphisms
M. Schneider (&)
Department of Marine Zoology, Ichthyology Section,
Senckenberg Research Institute and Natural History Museum,
Senckenberganlage 25, 60325 Frankfurt am Main, Germany
e-mail: mschneider@senckenberg.de
H. Sannæs H. Knutsen
Institute of Marine Research, Flødevigen Marine Research
Station, 4817 Arendal, Norway
P. E. Jorde
Centre for Ecological and Evolutionary Synthesis (CEES),
Department of Biology, University of Oslo, Blindern,
P.O. Box 1066, 0316 Oslo, Norway
Fishes of the gadiform family Macrouridae (grenadiers) are
primarily deep dwelling, benthopelagic species found in all
oceans. The majority of the more than 380 grenadier species known to date occur between 200 and 2,000 m depth
(Nelson 2006; Froese and Pauly 2008), but many species
are also found down to more than 6,000 m (Cohen et al.
1990). The carapine grenadier Coryphaenoides carapinus
is an abyssal species, and specimens were captured below
5,000 m depth in the South-eastern Atlantic during a cruise
of R/V Meteor in 2005. Two of the specimens were
obtained from Agassiz-trawls in the Cape Basin and a third
one in the equatorial Guinea Basin.
The genus Coryphaenoides is species-rich and includes the
largest growing and commercially most important species of
the family, such as the roundnose grenadier C. rupestris
inhabiting depths down to 2,200 m in the North Atlantic.
Coryphaenoides rupestris was harvested commercially in the
1970s with up to 82,000 t year-1, declining to 32,000 t in
2005, and the species is currently facing overexploitation
(FAO, http://www.fao.org/fishery/). Other representatives of
the family, which are targeted actively by fisheries, are found
in particular in the genus Macrourus (e.g., M. berglax in the
North Atlantic, and M. carinatus and M. holotrachys in the
South Atlantic; Cohen et al. 1990). All these species are poorly
known with respect to population structure, and the development of molecular markers should provide an important tool
for stock identification and fishery management. Microsatellites will further help to understand distribution and dispersal
patterns in the huge dimensions of the abyssal plains.
Here we report for the first time the isolation and
characterisation of eleven microsatellite loci for the abyssal
carapine grenadier Coryphaenoides carapinus Goode and
Bean 1883 and their cross-amplification in the two related
species C. rupestris Gunnerus 1765 and Macrourus berglax
Lacépède 1801.
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Total genomic DNA was extracted from gill tissue of
three specimens of C. carapinus. Genomic DNA was also
extracted from muscle tissue of nine specimens from each of
two related species, C. rupestris and M. berglax, sampled by
commercial fishing vessels at Rockall and on the east coast of
Greenland, respectively. Extraction was done according to
manufacturer’s protocol, using the Viogene Blood and Tissue Genomic DNA Extraction Miniprep System (Viogene
Inc.). Genetic Identification Services (GIS, Inc., Chatsworth,
CA, USA; http://genetic-id-services.com/) was contracted to
develop and screen four libraries enriched for tetranucleotide
(AAAC), (CATC), (TACA), and (TAGA) motifs, following
their proprietary protocol (Meredith and May 2002;
Schwartz and May 2004). For the successful libraries, a total
of 100 clones were sequenced and 11 primer pairs were
designed. All 11 loci amplified reliably, and all further tests
were restricted to these loci. Microsatellites were amplified
by polymerase chain reaction (PCR) in 10 ll reaction volumes. The specific primers (Sigma–Aldrich) were tagged
with a fluorescent dye (CY-5) at the 50 end of each forward
primer. The standard set of PCR conditions included 0.4 mM
dNTPs, 0.125 lM of each primer, 0.06 U/ll Eppendorf 5
Prime Taq DNA Polymerase and self-adjusting 109
magnesium buffer, included in the Eppendorf kit. The
maximum capacity of the buffer is up to 2.0 mM Mg2?
(Eppendorf AG, Germany). Dilution was done using
Eppendorf Molecular Biology Grade Water. Each well was
added 1 ll of template DNA extracts, corresponding to
25–50 ng of DNA. Amplifications were performed using a
BIORAD MYCycler thermal cycler, with PCR program as
follows: An initial denaturation step at 95°C for 5 min, followed by 30 cycles of denaturation, annealing and synthesis,
each for 30 s at temperatures 95, 56, and 72°C, respectively,
and finally one 15 min elongation step at 72°C. Note that
optimal annealing temperatures differ somewhat among
primer pairs (cf. Table 1), but we chose a common temperature (56°C) to simplify the laboratory protocol. Allele sizes
and genotypes were determined by fragment analysis, using a
Table 1 Summary of primer characteristics of 11 microsatellite loci
in the carapine grenadier (Coryphaenoides carapinus), giving GenBank accession numbers (GenBank), optimum annealing temperature
(Ta), repeat motifs in which ‘‘N’’ refers to miscellaneous nonrepetitive sequence of varying length, and forward (F) and reverse (R)
primer sequences
Locus/GenBank
Ta (°C)
Repeat motif
Primer sequence (50 –30 )
Size range (bp)
NA
HE
HO
CaraA8
57.5
(GTTT)6N(TCAA)11
F: TCGGGCTTAGAAAGTGCTG
198–214
3
0.73
0.33
0.60
-0.09
FJ694860
FIS
R: TCAGGACCACCTCCAGAAC
CaraA10
55.8
(GTTT)6
F: CCTCTCTCCTTACGTGGAGTTC
123–139
5
0.93
1.00
FJ694861
CaraA102
57.5
(GTTT)8
R: TGAGCACAACTGTAAACACAAC
F: CTTGCCTTGTTGGATGATG
252
1
0
0
–
NA
–
–
–
–
150–198
4
0.80
0.67
0.20
224–250
5
0.93
0.67
0.33
208–242
3
0.83
0.50
0.50
162–176
2
0.50
0.50
0.00
297–330
5
0.93
0.67
0.33
R: CGTAATGTGGCTGTGTGC
FJ694862
CaraA106a
56.2
(CA)45
CaraA109
57.1
(CA)10N(AAAC)7
58.0
(CCAT)12
FJ694865
CaraB114
56.7
(CCAT)8
F: CCACCTGCTTCAGCCTAT
R: AGGAGGAATTAGAGAGTGTGG
56.1
(CCAT)6
FJ694867
CaraC1
F: CCTCTCATCAGCACCACTGT
R: ACTGGAACGCACCATAACTG
FJ694866
CaraB116
F: CCTTGCCTCACACCTCTG
R: CCGTTCTTGCTTCTGTATGC
FJ694864
CaraB1
F: GGCTATTTAACCACACGCTTAC
R: CGGAAAAGTTGCATTAGAGG
FJ694863
F: CGACAATGACAGGTTCATG
R: CAGGAGGAACTGGAGAGTG
58.3
(TACA)7
F: TGCCCTTAGTGACAATCCAC
R: AGCACATTCTCTGGTTTACTGC
FJ694868
CaraC7
FJ694869
56.2
(TACA)11N(CA)14
F: GCTGGTGGTCAAGTGAATC
R: GCATTGGCTGTATTGTGC
164–188
4
1.00
1.00
0.00
CaraC117a
57.9
(GTAT)10
F: GGTGGAGGGATCATAAACAAG
255–311
3
0.80
0.00
1.00
0.75
0.53
0.35
FJ694870
Average
R: CATGCCTGAATAACGACTGAC
Size range of fragments (bp), number of alleles (NA), expected (HE) and observed (HO) heterozygosities, and deviation (FIS) from HardyWeinberg expectations (HWE) are based on a sample of three individuals. No significant deviations from HWE were observed. ‘‘NA’’ means no
useful amplification. Note that the locus CaraA106a did not amplify on C. carapinus, but did so on Macrourus berglax (cf. Table 2)
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Table 2 PCR cross-amplification of microsatellite loci in other macrourid species using 11 primers developed for carapine grenadier (Table 1)
Locus
C. rupestris (n = 9)
M. berglax (n = 9)
Size range (bp)
NA
HE
HO
FIS
Size range (bp)
CaraA106a
NA
–
–
–
–
85–107
4
0.67
0.67
0.01
CaraA109
188–202
4
0.66
0.44
0.34
NA
–
–
–
–
CaraC7
132–170
10
0.90
0.88
0.03
210–240
9
0.88
0.89
-0.01
0.78
0.66
0.16
0.78
0.78
0.00
Average
NA
HE
HO
FIS
Two of the 11 primers successfully amplified and were found to be polymorphic among nine individuals (n = 9) from each of roundnose
grenadier (Coryphaenoides rupestris) and onion-eye grenadier (Macrourus berglax). There was no useful amplification from the remaining eight
loci (not shown). Size range (in base pairs, bp) refers to specific alleles, NA is total number of alleles, HE refers to expected and HO to observed
heterozygosities, and FIS to deviation from Hardy-Weinberg expectations (HWE). No significant deviations from HWE were found. ‘‘NA’’ refers
to non-amplified loci
capillary sequencer (Beckman Coulter CEQ 8000) and CEQ
8000 Genetic Analysis Software (v8.0) (http://www.
beckman.com/products/specifications/geneticanalysis/ceq/
default.asp). Data analysis was performed using the software GENEPOP on the web (http://genepop.curtin.
edu.au/) and Genetic Data Analysis (v1.0; Lewis and
Zaykin 2001).
Nine polymorphic loci could be identified in C. carapinus (Table 1), segregating for 2–6 alleles (mean 4)
among the three scored individuals. The proportion of
heterozygous individuals (HO) among the nine loci ranged
from 0.0 to 1.0, with a mean of 0.53. No significant deviations from Hardy-Weinberg genotype proportions were
found, but the low number of sampled individuals for this
species rends these test results largely uninformative. Tests
for potential linkage disequilibrium among loci could not
be performed because of the sparseness of the contingency
tables (each two-locus genotype combination was observed
in one copy only).
We cross-tested all microsatellite loci on two other
macrourids: roundnose grenadier C. rupestris (n = 9) and
onion-eye grenadier M. berglax (n = 9). Three of the
eleven primers successfully amplified at two polymorphic
loci in each species (Table 2). In C. rupestris four and ten
alleles were observed at each locus among the nine sampled individuals, with HO of 0.44 and 0.88, respectively. In
M. berglax two loci segregated for four and nine alleles
with HO of 0.67 and 0.89, respectively.
Acknowledgments We thank Michael Türkay and Kristin Pietratus
(Senckenberg Research Institute) for taking the tissue samples of
Coryphaenoides carapinus onboard of R/V Meteor during DIVA 2
cruise. We thank Kate Enersen (IMR) for laboratory assistance. This
study was partly funded by a network grant in the EuroDEEP-programme (BIOFUN-DEECON) of the European Science Foundation
and by a research grant of the Deutsche Forschungsgemeinschaft
(DFG, KR-1758/2-1). The investigations are element of the
CeDAMar and Mar-Eco of the ‘‘Census of Marine Life’’-initiative.
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