Molecular Ecology Notes (2005) 5, 463–465
doi: 10.1111/j.1471-8286.2005.00883.x
PRIMER NOTE
Blackwell Publishing, Ltd.
Isolation and characterization of microsatellite loci in Pseudoplatystoma corruscans (Siluriformes: Pimelodidae) and cross-species amplification E L O I S A R E V A L D A V E S , L U I Z H . G . P E R E I R A , F A U S T O F O R E S T I and C L A U D I O O L I V E I R A Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista, 18618–000, Botucatu, SP, Brazil
Abstract A total of five polymorphic microsatellites loci from Pseudoplatystoma corruscans were isolated and characterized. A population survey involving 43 specimens resolved a large number of alleles (range seven to eight among loci) and high observed heterozygosity (0.500 –0.615), indicating its usefulness in population genetics studies. Cross-species amplification was successful in four other Pimelodidae species. Keywords: catfish, cross-amplification, microsatellites, Pseudoplatystoma Received 30 September 2004; revision accepted 18 November 2004
The Neotropical freshwater fish fauna includes several large catfishes that are very important in commercial fisheries, many of them belonging to the family Pimelodidae, including those in the genera Pseudoplatystoma, Brachyplatystoma, Phractocephalus, and Zungaro (Ferreira et al. 1998). Fishes of the genus Pseudoplatystoma have an important ecological role as predators in the ecosystems where they occur (Miranda 1997). These species exhibit extensive migratory movements during their life cycles which are being affected by power plant construction. The environmental changes associated with water pollution and fisheries have heightened conservation concern for these species. Several efforts have been made trying to protect the species in national parks and propagate these species in captivity (Miranda 1997) to produce fishes for markets and also to release the reared fish to enhance natural populations. To further genetic population analysis of natural and hatchery populations, we developed microsatellite primers for one species, Pseudoplatystoma corruscans. Microsatellite loci were isolated from a partial library enriched for dinucleotide loci, following the protocols of Hamilton et al. (1999). Approximately 10 µg of DNA from one P. corruscans individual was extracted using the phenolchloroform method as described by Sambrook & Russel (2001). The DNA was digested with DraI and SspI and electrophoresed in 2% low-melting agarose gel. DNA fragments ranging from 400 to1200 bp were excised from the gel, Correspondence: C. Oliveira, Fax: 55 14 38153744; E-mail:
[email protected] © 2005 Blackwell Publishing Ltd
purified with phenol-chloroform, and ligated into the adaptors Snxr 5′-P-GCTTCTGCTAGCAAGGCCTTAGA3′ and Snx 5′-CTAAGGCCTTGCTAGCAGAAGC-3. Enrichment was performed using biotinylated (AC)18 and (AG)18 oligonucleotides and streptavidin-coated magnetic beads (Streptavidin Magnesphere Paramagnetic Particles, Promega). The recovered DNA was amplified by using the Snx adaptors as primers. Amplified fragments were ligated into the pGEM-T (Promega) and then used to transform DH5α Escherichia coli cells (Subcloning Efficiency DH5α Competent Cells, Invitrogen). Plasmid DNAs were isolated using phenol-chloroform and sequenced with an ABI Prism 377 automated sequencer using the BigDye Terminator kit (Applied Biosystems). Flanking primers were designed using the primer 3 software (Rozen & Skaletsky 1998). A total of 81 positive colonies were isolated, 22 of which were sequenced. Of these, 16 sequences (GenBank Accession nos AY737063 to AY737078) resulted in good quality flanking sequences and uninterrupted repeat motifs and were selected for further characterization. The analysis were conducted with a sample of 43 fishes collected in the Taquari River, Mato Grosso do Sul, Brazil. Polymerase chain reactions (PCRs) were performed in a 10 µL reaction volume containing approximately 10 ng of DNA, 1× PCR buffer, 0.3 µm of each primer, 1.5 mm MgCl2, 0.25 U Pht Taq and water. The conditions for amplification were 2 min at 95 °C followed by 30 cycles of 5 s at 95 °C, 10 s at the annealing temperature (see Table 1) and 10 s at 72 °C with a final extension time of 10 min at 72 °C.
464 P R I M E R N O T E Table 1 Characteristics of five polymorphic and three monomorphic loci for Pseudoplatystoma corruscans Locus/ Accession no. Pcor1 AY737063 Pcor2 AY737064 Pcor5 AY737067 Pcor6 AY737068 Pcor7 AY737069 Pcor8 AY737070 Pcor10 AY737072 Pcor21 AY737078
Repeat motif
Primers (5′–3′)
(TC)9GC(TC)9
F: AAACCCGAGGATAACCAGTC R: CAGCGTGCTACTAACACAAAC F: GATATGCAAATAAGAAGGTC R: TCTTCTGGCTTTTCCTCCTCT F: GACTAAGATTACACAGAGATTC R: CTTGGTGGGGAAACAGGC F: CATGATTAAGGGCTGTAT R: GGGATAGATGGCGTAAGCAG F: ATGCTGGGATACGCTCAGAC R: GTGGCGAGTGAACAAGTCC F: ACACCATACGCACACACTCG R: TGAGGTCGGGTGATAAGGTC F: TTTAAGACAGCACAGCCTGTGGGG R: AAGACAGCGCCATAGAGTTCTGCC F: TCACCGAGAGGTCTGACCATGA R: CTGTGGTTAACCAGCTAGCAC
(AG)19 (TC)8CC(TC)15 (TG)5(TA)5 (AG)8GG(AG)2CG(AG)4 (AC)12 (GTCG)15(GT)9CC (GT)13
Ta (°C)
Cloned allele size
n
55
105
42
55
199
55
A
HO
HE
HWE
7
0.500
0.734
**
43
12
0.605
0.848
**
155
42
9
0.595
0.725
NS
56
156
42
1
—
—
—
56
209
42
1
—
—
—
56
171
42
1
—
—
—
49
173
39
23
0.615
0.942
**
55
117
42
8
0.500
0.820
**
Ta, annealing temperature; n, sample size; A, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity. Hardy– Weinberg equilibrium (HWE); NS, not significant; **P < 0.01. Table 2 Cross-amplification data of 10 additional siluriform taxa. n, sample size; P, polymorphic; M, monomorphic; —, no amplification Locus Species
n
Pcor01
Pcor02
Pcor05
Pcor06
Pcor07
Pcor08
Pcor10
Pcor21
Pimelodidae Pseudoplatystoma fasciatum Steindachneridion scripta Pirinampus pirinampu Pimelodus maculatus
8 8 3 3
P P P P
P P P P
P P P P
M M M M
P M — —
P M P P
P P P P
P M P —
Pseudopimelodidae Lophiosilurus alexandri Pseudopimelodus mangurus
4 5
M P
M M
M M
M M
— —
M M
— —
— —
Heptapteridae Imparfinis mirini
5
—
M
—
M
—
M
—
—
Cetopsidae Cetopsis coecutiens
5
P
P
M
—
M
M
—
M
Loricariidae Neoplecostomus paranensis
5
P
M
M
M
—
M
—
—
Callichthyidae Hoplosternum littorale
5
M
M
M
M
M
M
—
—
Among the 16 tested primer pairs, eight showed clear band patterns. Five loci were highly polymorphic (Table 1) and three were monomorphic (Pcor6, Pcor7, and Pcor8). The number of alleles at each polymorphic locus, their size range, and observed and expected heterozygosities are shown in Table 1. They were calculated using popgene version 1.31 (Yeh & Boyle 1997). Tests for linkage disequilibrium and deviations from the Hardy–Weinberg equilibrium (HWE) were performed using genepop (Raymond & Rousset 1995). The five polymorphic loci displayed from 7 to 23 alleles/
locus (mean across loci was 11.8) and the mean expected heterozygosity was 0.814. Of the five loci, all but one (Pco05) displayed significant deviations from Hardy– Weinberg expectations (P < 0.05). These deviations might be results of null alleles, nonrandom sampling, mating systems or inbreeding. None of the 10 pairs of loci compared exhibited significant genotypic disequilibrium (P < 0.05). Cross-species amplification was investigated in 10 additional siluriform fishes from six families (Table 2). A high © 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 5, 463–465
P R I M E R N O T E 465 level of cross-species amplification was observed among the species of Pimelodidae, but only a few primers produced results in other families analysed (Table 2), suggesting that the isolated loci are largely characteristic of Pimelodidae. Our results suggest that these microsatellites are suitable for population genetic analysis and for parentage studies in P. corruscans. The generality and causes of the observed deviations from HWE, however, will need to be resolved before the loci can be widely used in population studies, to design management programs in a hatchery population, or be applied to other cultivated catfish species.
Acknowledgements We thank José A. Senhorini, Priscila Gusmão-Pompiani, and Fernando Pompiani for help with fish collection. We appreciate the comments on our manuscript made by Eric B. Taylor. Financial support for this study was provided by CNPq and FAPESP.
© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 5, 463–465
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