Polymorphic microsatellite loci in the black-and-gold chromis, Neoglyphidodon nigroris (Teleostei: Pomacentridae)

The black-and-gold chromis, Neoglyphidodon nigroris, is a highly territorial fish which is common in coral-rich lagoons and forereef habitats throughout the Indo-West Pacific (Froese & Pauly 2003). Neoglyphidodon nigroris deposits benthic eggs in its territory but it is not known whether the juveniles are philopatric or widely dispersed (e.g. to reduce competition between kin). Data on dispersal extent and how adjacent reefs are linked would significantly aid in understanding how management efforts are protecting the genetic integrity of these increasingly exploited ecosystems. As part of an effort to better understand the genetic relationships between N. nigroris individuals on reefs and also to gain an insight into the dispersal of this species between reefs we developed a panel of microsatellite loci using an enrichment procedure based on the protocol of Gardner et al. (1999).

We constructed a partial genomic library that was enriched for CA motifs using genomic DNA of a single adult damselfish. DNA was extracted from muscle tissue using a high-salt protocol (Aljanabi & Martinez 1997). Sau3AI (40 U; Boehringer-Mannheim) was used to digest approximately 10 µg of total genomic DNA which was ligated to 50 pmol of phosphorylated linkers (S61 5′-GGCCAGAGACCCCAAGCTTCG-3′ annealed to S62 5′-PO₄-GATCCGAAGCTTGGGGTCTCTGGCC-3′; Refseth et al. 1997). DNA fragments between 500 and 1000 bp were excised from a 2% NuSieve GTG agarose TAE gel (FMC Bioproducts) and purified using a QIAquick gel extraction kit (Qiagen). For enrichment we used 1 mg of M2-80 streptavadin-coated magnetic beads (Dynal) incubated with 200 pmol of 3′-biotin-labelled (CA)₁₂ oligonucleotide (MWG Biotech). After a series of differential stringency washes in 2× SSC and 1× SSC, the enriched DNA was recovered, made double stranded and amplified in a 25-µL polymerase chain reaction (PCR) [75 mm Tris-HCl, 20 mm (NH)₄SO₄, 0.01% (v/v) Tween 20, 0.2 mm each dNTP, 1.5 mm MgCl₂, 250 pmol primer S61 and 1.25 U of Taq polymerase (ABgene)]. The thermal profile of the PCR was 5 min at 95 °C, 25–30 cycles of 50 s at 95 °C, 1 min at 56 °C and 2 min at 72 °C followed by 72 °C for 10 min. The DNA was purified using a QIAquick PCR purification kit (Qiagen), ligated into pGEM®-T vector (Promega) and then transformed into JM109 Escherichia coli competent cells (Promega). Recombinant clones were identified using black/white screening on S-gal (Sigma) agar/ampicillin plates. Plasmids containing a microsatellite insert were identified by two or more amplified products after PCR primed with 50 pmol SP1 and 25 pmol (GT)₁₂ oligonucleotide (see Gardner et al. 1999). Positive clones were cycle sequenced using Big Dye™ chemistry (PE Applied Biosystems) and electrophoresis on an ABI377. Full details of the enrichment protocol are provided by Bloor et al. (2001). Primers flanking the repeat regions were designed using primer2 (S.J. Kemp, unpublished). We identified 85 putative positive clones from 288 colonies. Thirty-two positive clones were sequenced, yielding 28 separate microsatellites with suitable flanking sequence for primer design (three pairs of sequences were identical and primers could not be designed at one locus). Eleven of the remaining primer sets showed either inconsistent PCR amplification or multiple bands.

The remaining loci were tested for polymorphisms using DNA extracted from pelvic fin clips of 16 N. nigroris from the two forereef sites off Hoga island, southeast Sulawesi, Indonesia. Microsatellite alleles were amplified by PCR in a 10-µL reaction volume on a Dyad DNA engine (MJ Research Inc.). The PCR conditions were 1 min at 95 °C, six cycles of 30 s at 95 °C, 30 s at T_a and 45 s at 72 °C, 26 cycles of 30 s at 92 °C, 30 s at T_a and 55 s at 72 °C followed by 72 °C for 30 min (T_a is the annealing temperature at each locus; Table 1). Each reaction contained 75 mm Tris-HCl, 20 mm (NH)₄SO₄, 0.01% (v/v) Tween 20, 0.2 mm each dNTP, 1.5–3.5 mm MgCl₂ (Table 1), 10–50 ng template DNA, 10 pmol each primer and 0.25 U of Taq polymerase (ABgene). The forward primers were 5′ labelled with either 6-FAM, NED, PET or VIC fluorescent dyes (Applied Biosystems) for detection (Table 1). The PCR products were pooled with a genescan 500 bp (LIZ) size standard (Applied Biosystems) and separated by capillary electrophoresis through a denaturing acrylamide gel on an ABI3100 automated sequencer (Applied Biosystems). We used arlequin version 2.001 (Schneider et al. 2000) to calculate observed (H_O) and expected (H_E) heterozygosities and linkage disequilibrium between all pairs of loci.

Two loci were monomorphic and 15 loci were variable and resolved distinct alleles within the expected size range. The observed number of alleles varied from two up to 23, H_O varied between 0.13 and 1.00 whilst H_E ranged between 0.24 and 0.98 (Table 1). Fourteen of the 105 possible pairwise comparisons between polymorphic loci showed significant linkage disequilibrium [after a sequential Bonferroni correction (Rice 1989) to maintain a type I error rate of α = 0.05 for each locus]. Four of the loci (LIST12-006, LIST12-007, LIST12-016 and LIST12-025) did not demonstrate significant linkage disequilibrium with any other locus, whilst LIST12-002b and LIST12-023 were responsible for the majority of the significant departures from linkage equilibrium (see Table 2). These markers will allow us to discriminate between closely related individuals and also to assess the connectedness of different populations of the black-and-gold chromis.