Antoni et al: Microsatellite markers for Thunnus atlandcus 
323 
procedure. In our study, this method was applied to 
rapidly identify microsatellite loci in the blackfin tuna. 
Materials and methods 
Genomic DNA from fin tissue of one blackfin tuna spec- 
imen collected offshore of the Louisiana coastline was 
extracted by using a standard phenol-chloroform pro- 
tocol (Sambrook et al., 1989). An Illumina 1 paired-end 
library, which allows sequencing of both ends of DNA 
fragments, was prepared and sequenced on an Illumina 
HiSeq 2000 platform (Illumina, San Diego) according 
to methods described by Castoe et al. (2012). Reads 
were quality controlled and trimmed for low-quality 
data (phred scores <30 ). In the program PAL_FINDER, 
vers. 0.02.03, a Perl script developed by Castoe et al. 
[2012] and available at http://sourceforge.net/projects/ 
palfinder/), 5,874,294 reads were screened for microsat- 
ellite arrays that contained a minimum of 12 repeats. 
Use of a minimum of 12 repeats, in our experience, en- 
sures that the selected microsatellite loci are likely to 
be polymorphic. The search in PAL_FINDER was con- 
tinued until 45 dinucleotide loci and 5 tetranucleotide 
loci were discovered, requiring screening of 286,240 
reads and the whole database (5,874,294 reads) for di- 
nucleotides and tetranucleotides, respectively. 
Specific polymerase chain reaction (PCR) primers 
were designed with the open-source software Primer3 
(Untergasser et al., 2012; Koressaar and Remm, 2007; 
source code available at http://sourceforge.net/projects/ 
primer3/). The designed primers were tested for ampli- 
fication success and scoring reliability and then evalu- 
ated at different annealing temperatures using blackfin 
tuna samples provided by the Louisiana Department of 
Wildlife and Fisheries. Samples had been collected off- 
shore of the Louisiana coastline during the spring of 
2013. Amplification success of the candidate loci during 
PCR was tested by assaying 6 specimens. PCR reac- 
tions were performed in a total volume of 5.6 pL that 
contained 7-13 ng of genomic DNA, 2.2 pmol of each 
primer, 1.1 nmol of dNTPs (Promega Corp., Madison, 
WI), 8.4 nmol of MgCl 2 (Promega), 0.28 U of Taq poly- 
merase (Promega), and IX of buffer (Promega). Ampli- 
fication by PCR consisted of an initial denaturation at 
95°C for 5 min, 35 cycles of 95°C for 30 s, annealing 
temperature (T a ) for 30 s, 72°C for 45 s, and a final 
extension of 15 min at 72°C. T a was 62°C for the first 
7 cycles, 60°C for the following 7 cycles, and 56°C for 
the remaining 21 cycles. The obtained PCR products 
were evaluated through electrophoresis on high-reso- 
lution NuSieve GTG Agarose gels (Lonza Group, Basel, 
Switzerland). 
Loci that showed consistent amplification success 
and polymorphic PCR products were selected for fur- 
1 Mention of trade names or commercial companies is for iden- 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA. 
ther testing, and the 5' end of 1 of the 2 primers was 
labeled by using one of the fluorescent dyes 6-Fam, 
Hex, or Ned to allow detection and scoring on an auto- 
mated DNA sequencer. Evaluation of scoring reliability 
of the tested loci was based on DNA samples from 8 
blackfin tuna specimens. The optimal T a for amplifica- 
tion of each locus was then determined during PCR re- 
actions by using DNA from 4 blackfin tunas, and was 
carried out at 6 annealing temperatures held constant 
through the 35 amplification cycles and ranging from 
52°C to 62°C. PCR products were run on an ABI Prism 
377 96-lane DNA Sequencer (Applied Biosystems, Life 
Technologies, Carlsbad, CA). The obtained electrophe- 
rograms were analyzed with Applied Biosystems Ge- 
neScan software, vers. 3.1.2 (Life Technologies), and 
alleles were scored in Applied Biosystems Genotyper 
software, vers. 2.5 (Life Technologies). The polymorphic 
loci that could be scored reliably were characterized 
statistically on the basis of the genotypes of 35 black- 
fin tuna specimens provided by C. Pau and L. Reynal, 
both of IFREMER, La Martinique. Specimens had been 
caught offshore of the island of Martinique in the sum- 
mer of 2013. 
Results 
Amplification tests were conducted on 50 primer pairs, 
45 potentially amplifying dinucleotide microsatel- 
lites, and 5 amplifying tetranucleotide microsatellites. 
Twenty-four loci were amplified consistently across the 
tested specimens and were all labeled with fluorescent 
dyes for further evaluation and optimization of anneal- 
ing temperature. Twenty loci (19 dinucleotides and 1 
tetranucleotide) gave scorable PCR products and are 
described in Table 1. The number of alleles (A), expect- 
ed heterozygosity (H ), and inbreeding coefficient (Fig) 
were calculated with the software FSTAT, vers. 2. 9. 3. 2 
(Goudet, 1995). Per locus estimates ranged from 5 to 
26 for A, from 0.640 to 0.969 for H and from -0.003 
e> 
to 0.268 for Fig. Conformance of genotypic proportions 
to Hardy-Weinberg (H-W) equilibrium expectations was 
tested with exact tests in the software GENEPOP, vers. 
4.1 (Raymond and Rousset, 1995; Rousset, 2008). Geno- 
typic proportions did not depart significantly from H-W 
equilibrium expectations, except for the loci BT6, BT27, 
BT47, and BT91. The departure at locus BT27 was not 
significant after Bonferroni correction (Rice, 1989). 
Analyses in Micro-Checker, vers. 2.2.3 (Van Oosterhout 
et al., 2004) revealed possible occurrence of null alleles 
at loci BT6, BT47, and BT91. There was no evidence of 
scoring artifacts at locus BC27. 
Discussion 
On the basis of the significant departure of genotypic 
proportions from Hardy-Weinberg equilibrium expec- 
tations and the inference of null alleles at loci BT6, 
