Microsatellite multiplexes for high-throughput genotyping of French grunts (Haemulon flavolineatum, Pisces: Haemulidae) and their utility in other grunt species

The genus Haemulon (grunts) contains 18 species, 13 of which are found in the Western Atlantic (Lindeman 1986). Grunts are some of the most abundant western Atlantic reef fish and are an important component of regional fisheries (Lindeman 1986). Some grunt species face threats from a variety of sources, including over-fishing, high mortality of juveniles from shrimp trawls, and destruction of nursery habitats (Lindeman 1986; Lindeman et al. 2000, 2001; Nagelkerken et al. 2002). Conservation efforts for these and other Caribbean reef fish will benefit from an understanding of population structure and the extent of dispersal among populations (Cowen et al. 2000).

Grunts begin life as planktonic larvae that eventually settle onto the reef as juveniles. As adults, grunts are relatively sedentary, making short daily migrations from patch reefs to surrounding sea grass beds to feed (Ogden & Ehrlich 1977). As a consequence of their behaviour, long-distance dispersal between reefs may only occur by the transport of planktonic larvae by ocean currents. Two characteristics of larval grunts, however, make it likely that most are retained near their natal reef (Lindeman et al. 2001): larval duration in grunts is relatively short (mean 14 days) and larvae tend to remain inshore and close to the bottom.

We developed microsatellite loci for French grunts to assess the importance of dispersal for population structure in this species. We optimized these loci into three polymerase chain reaction (PCR) multiplexes to facilitate high-throughput genotyping, then tested the suitability of these loci for other Caribbean grunt species.

Genomic DNA for library construction was extracted from fin tissue using a standard phenol–chloroform extraction. DNA was digested with DpnII and fragments 400–1000 bp in length were cloned into Lambda Zap Express (Stratagene) (Hughes & Moralez DeLoach 1997). We screened 180 000 colonies with the oligos AAT₁₀ and AAC₁₀. We sequenced 28 positive clones using ABI Big Dye Terminator Cycle Sequencing vs. 3.0 chemistry (PE Biosystems). Sequences were analysed on an ABI 310 Genetic Analyser (PE Biosystems). The oligo AAC₁₀ did not hybridize to any sequence containing this repeat motif and instead hybridized to 13 clones containing (AC)_n dinucleotide repeats. We designed primers for 13 clones that contained ≥ 8 uninterrupted trinucleotide repeats and ≥ 10 dinucleotide repeats using Oligo® 4.04 (Rychlick 1992).

DNA for genotyping was extracted from fin tissue (preserved in 20% dimethylsulfoxide, 6 m NaCl) using the single tube method of Estoup et al. (1996). PCR reactions (10 µL) contained 100 ng DNA, 50 mm KCl, 10 mm Tris-HCl pH 9.0, 1.5 mm MgCl₂, 0.1% Triton X-100, 200 µm of each dNTP, 0.2 U Taq DNA polymerase (Promega), and 0.5 µm of each primer. Reactions were cycled using a Hybaid Touch Down thermal cycler using the ‘simulated tube’ function. Cycling parameters were: one cycle at 92 °C for 60 s, followed by 30 cycles of 5 s at 92 °C, 5 s at annealing temperature, and 10 s at 72 °C, and then a final extension at 72 °C for 2 min. Optimal annealing temperatures and suitability of loci was initially determined using nondenaturing 6% polyacrylamide gel electrophoresis (PAGE) and visualized using ethidium bromide.

Eleven loci were chosen for their polymorphism and ease of scoring, and one primer of the pair was labelled with a fluorescent dye (Table 1). Genotypes were scored using the ABI 3730 XL Genetic Analyser and the software genemapper 3.0 (PE Biosystems). To determine multiplex PCR conditions, we combined loci based on annealing temperature and allele size range determined from PAGE. Four individuals were then screened using the above PCR conditions with the exception that all primer concentrations were lowered to 0.1 µm to decrease nonspecific amplification and competitive interactions among primers (Neff et al. 2000). Based on these tests, primer concentrations were then increased or decreased (0.05–0.25 µm) for specific loci and Taq DNA polymerase was increased to 0.5 or 1.0 U per reaction to give consistent amplification across loci. We also found that decreasing the annealing temperature by 5 °C greatly improved amplification success of some multiplexes. We then screened 16 individuals across populations to determine the consistency of amplification. Often primer concentrations had to be slightly altered to take into account the larger variation in template quality and quantity among these individuals. We were able to group the loci into three multiplexes (A–C) (Table 1). Annealing temperatures and Taq DNA polymerase concentrations (units per reaction) for the multiplexes were: multiplex A: 50 °C and 1 U Taq; B: 55 °C and 1 U Taq; and C: 55 °C and 0.5 U Taq. We also tested these loci separately on four other grunt species (Table 2). The loci work especially well in H. aurolineatum, H. plumieri and H. sciurus, and may allow population genetic studies of these species without further primer development.