Molecular Ecology Notes (2004) 4, 551–553
doi: 10.1111/j.1471-8286.2004.00729.x
PRIMER NOTE
Blackwell Publishing, Ltd.
Isolation of highly polymorphic microsatellite loci from the temperate damselfish Parma microlepis B E L I N D A G . C U R L E Y * and M I C H A E L R . G I L L I N G S † *School of Marine Biology and Aquaculture, James Cook University, Townsville, Queensland, Australia 4811, †Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia 2109
Abstract Microsatellites were isolated from the damselfish Parma microlepis (Günther 1862) (Pomacentridae) and screened for 100 individuals. Seven of the eight loci tested were highly polymorphic, having 14–43 alleles with average heterozygosities between 0.86 and 0.97. These loci should be informative for studies on population genetics of this species. Keywords: connectivity, genetic diversity, microsatellites, Parma microlepis, Pomacentridae, temperate reefs Received 18 March 2004; revision received 28 May 2004; accepted 28 May 2004
The temperate damselfish Parma microlepis is endemic to rocky reefs of southern Australia (Hutchins & Swainston 1986) and is one of the most abundant reef fishes in the Sydney region (Curley et al. 2002). Parma microlepis is territorial with limited postsettlement movement (< 100 m2) (Moran & Sale 1977; Kingsford & Gillanders 2000). Dispersal among reefs is therefore achieved via the pelagic larval phase (Tzioumis & Kingsford 1995). Knowledge of dispersal capabilities, coupled with its relative abundance, makes P. microlepis an ideal candidate to test models of gene flow and connectivity among reef fish populations. This information is fundamental to conservation and fisheries management. Here we describe the characterization of microsatellite loci from P. microlepis to be used in this context. One hundred specimens were collected from two locations in coastal New South Wales, Australia. Fin clips were stored in 70% ethanol. DNA for a genomic library was extracted from frozen liver of two individuals using phenol/chloroform/SDS extraction (Gillings & Fahy 1993). Approximately 3 µg genomic DNA was digested to completion with RsaI or HinfI. Fragments of 300 –900 bp were purified from agarose gels using Wizard SV gelpurification columns (Promega). Size fractions were pooled and ligated into SmaI digested, phosphatase treated pUC18 (Amersham Pharmacia Biotech) using Promega T4 DNA ligase. Ligations were used to transform TOP-10 compeCorrespondence: Associate Professor Michael Gillings. Fax: 61 29850 9237; E-mail:
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tent E. coli JM109 (Invitrogen), which were plated to LB agar containing ampicillin (100 µg/mL), IPTG and X-gal. Recombinant bacterial colonies (1125) were picked into Nunc 384 well microtitre trays, incubated overnight in LB broth, and stored at −80 °C in 15% glycerol. A pin replicator was used to transfer colonies to nitrocellulose membranes laid over LB agar, and incubated overnight. Cells were lysed by layering the membranes on 1 m NaOH (2 × 10 min) and neutralized in 1 m Tris-HCl (2 × 10 min). DNA was fixed to the membrane using a Stratgene UV cross linker for 1 min. Oligonucleotide probes were 5′ labelled with [γ-32P] dATP (Perkin Elmer) using T4 polynucleotide kinase (Promega) and hybridized overnight at 50 °C in 6 × SSC with Easy Hyb (Boehringer). Filters were probed with (AC)10, and subsequently with a mixture of (AC)10(CT)10(GACA)5 and (AAAG)6. Filters were used to expose X-ray film for 2–12 h. Of 1125 clones, 39 responded to the (AC)10 probe (3.4%). An additional six colonies responded to the probe mixture. Plasmids were purified from 20 colonies that gave strong hybridization signals, using Promega Wizard columns, and DNA sequenced with the M13 primer (5′ GTAAAACGACGGCCAG) using the ABI Prism Big Dye terminator cycle sequencing kit (Applied Biosystems). Sequences were determined on an ABI377 system (Applied Biosystems) and edited using DNASIS (Hitachi) software. Eighteen of the 20 clones contained microsatellites in the sequenced region. Eight contained compound or multiple microsatellite regions. Two clones did not have sufficient flanking sequence for primer design. Primers were designed
552 P R I M E R N O T E Table 1 Microsatellite loci from Parma microlepis. Shown are the locus name, primer sequences, repeat motif, annealing temperature used (Ta °C), observed size range, number of alleles (k), observed heterozygosity (HO), and expected heterozygosity (HE), **statistically significant deviation from Hardy–Weinberg equilibrium (P < 0.001). Individuals genotyped (N) = 100 for each locus except PM2G2 where N = 99 Locus†
Primer sequences (5′– 3′)‡
Repeat
Ta °C
Size range
k
HO
HE
PM2N7
F: GAATGATGGAGCAGAAACAGG R: GGAAACTTTACACCGTGTCG fam F: TGTCAGTCGGATAGTACCTTCG R: TTTAAAGGCGGCTGATCTATC tet F: CGCTGTGAGTACTGATTCTGG R: GTCCAACAATACCCTGAGAGG fam F: AAGATGATCTCGAGGAGTGG R: CTGACAGGACAGAACACTTACC fam F: GCTGATGTGACAAACTGTTGC R: ATCACCAGGGATGCTTTACC hex F: TGTGTGTTTGCACCTGTTTG R: CCCTGCAATACCACTACAGC fam F: TAGAGCCCAGTCTCATCTCG R: GAGCTACCATTCCAGCACTCG hex F: CCACCTTTCTCAGCTCCTTCC R: CACAGCTCGCAGAGATGAAG tet
(GA)16
60
236–266
14
0.91
0.85
(ACAG)9(ATAG)21
60
135–239
20
0.94
0.92
(AC)19
60
111–155
20
0.90
0.84
(CA)5(CR)8(CA)24
58
330–422
36
0.97
0.97
(TG)19
58
127–171
21
0.95
0.93
(GT)13
62
121–159
16
0.86
0.88
(GT)54(CT)8
62
216–322
43
0.92**
0.97
(CA)7(CT)4
62
321–329
3
0.23
0.22
PM2G2 PM2D15 PM1M14 PM2G3 PM1E12 PM3L23 PM3K12
†GeneBank accession numbers for DNA sequences of loci are AY562367–AY562382. A further eight sequences useful for primer design are included in this deposition. Primer pairs used routinely in multiplex reactions were (G2 and D15) (M14 and G3) (E12, L23 and K12). ‡Primers were 5′ end labelled with the indicated fluorochrome.
for eight microsatellites using primer 3 (Rozen & Skaletsky 1998), setting the Tm between 58 and 62 °C. Primers were synthesized with 5′ fluorochrome labels (6FAM, HEX or TET) on the reverse primer (Sigma Genosys). Primer sequences, specific fluorochrome labels and empirically derived annealing conditions are given in Table 1. DNA was extracted from fin clips using a proteinase K/ salting out method (Sunnucks & Hales 1996). Microsatellite loci were amplified from 100 fish with a Hybaid Omne cycler using 50 ng of genomic DNA in 50 µL reactions. The concentrations of reagents in polymerase chain reactions (PCRs) were 2 mm MgCl2, 20 µg/mL RNAseA, 200 µm of each dNTP, 100 pmol of each primer, and 1 unit of Red Hot DNA polymerase (Advanced Biotechnologies) in the buffer supplied with the enzyme. PCR conditions were 94 °C for 3 min, followed by 35 cycles of 94 °C for 20 s, 58– 62 °C for 20 s (see Table 1), and 72 °C for 40 s, with a final extension at 72 °C for 10 min. An aliquot of each PCR was electrophoresed on 2% agarose and stained with ethidium bromide to check for amplification. PCRs were then diluted 1:10 in TE buffer and analysed on an ABI377 sequencer. Allele sizes were calculated using genescan and genotyper software (Applied Biosystems) in comparison to internal TAMRA labelled Gene-Scan 500 size standards (Applied Biosystems). Primer sets with different 5′ labels, nonoverlapping allele sizes and similar annealing temperatures were multiplexed to minimize time and costs.
All primer pairs generated scoreable polymorphisms at the microsatellite loci tested. Seven of the eight loci exhibited a large number of alleles (14–43) and very high observed heterozygosity (0.86–0.97). Hardy–Weinberg and linkage tests were conducted using genepop version 3.4 (Raymond & Rousset 1995), raising the default Markov chain parameters fourfold to account for the high heterozygosity. Locus PM3L23 exhibited a homozygote excess, possibly caused by preferential amplification of short alleles during multiplexing (Table 1). There was no significant linkage disequilibrium between loci. The loci described here should be valuable for studies on the population genetics of P. microlepis and may also be appropriate for use on related taxa.
Acknowledgements This research was funded by a James Cook University MRG awarded to MJ Kingsford, and a Macquarie University RDG awarded to MRG. BGC is the recipient of an Australian Postgraduate Award. We would like to thank Alan Curley for assistance in the field and Paul Worden and Marita Holley for technical assistance.
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© 2004 Blackwell Publishing Ltd, Molecular Ecology Notes, 4, 551–553
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