Description of novel microsatellite loci in the Neotropical fish Prochilodus argenteus and cross-amplification in P . costatus and P . lineatus

Prochilodus is one of the most important fish resources of South America, in addition to the important role it plays in nutrient cycling of Neotropical rivers. In the present study, we describe the isolation and characterization of nine novel microsatellite loci in Prochilodus argenteus. The number of alleles per polymorphic locus varied from 5 (Par76) to 21 (Par85), revealing a total of 116 alleles. The values of observed and expected heterozygosities ranged from 0.629 (Par69) to 0.926 (Par85 and Par86) and from 0.643 (Par66) to 0.931 (Par80), respectively. Furthermore, the ability of these and other previously described microsatellite markers to amplify orthologous loci was tested in two related species, Prochilodus costatus and Prochilodus lineatus. These loci will be useful for studies of population genetic structure in this group of fishes, and in aiding future genetic mapping studies of P. argenteus.

Family Prochilodontidae constitutes one of the most important fish resources of South America (Bayley and Petrere, 1989), in addition to its important role in nutrient cycling in Neotropical rivers (Flecker, 1996).Prochilodus comprises 49 nominal species of which only 13 species are valid (Castro and Vari, 2003).Among the species found in the São Francisco River basin, Prochilodus argenteus, popularly known as curimatã-pacu, forms a bulk of the subsistence fishery of the region, although numbers harvested have drastically declined in the last years (Sato and Godinho, 2004).
Microsatellites are polymorphic DNA sequences containing short tandemly arranged repetitions (Tautz, 1989), distributed throughout the genome (Litt and Luty, 1989), and found in all prokaryotic and eukaryotic genomes studied until now (Zane et al., 2002).Due to their high variability, these genetic markers have been widely used in genetic mapping (Knapik et al., 1998;Shimoda et al., 1999;Coimbra et al., 2003) and population structure studies (Hatanaka et al., 2006;Primmer et al., 2006).However, one of the great impediments for the wider use of microsatellites is the need to isolate and characterize these markers via cloning and sequencing of genomic libraries for each species of interest (Angers and Bernatchez, 1997).Nevertheless, once the flanking sequences of microsatellite markers are known, a large number of individuals may be rapidly genotyped.
Total genomic DNA was extracted from liver tissue of a P. argenteus individual using the phenol-chloroform method (Sambrook et al., 1989).A partial genomic library enriched for tetranucleotide loci was constructed following the protocols of Hamilton et al. (1999).The DNA was digested with BstU I and DNA fragments ranging from 300 to 1200 bp were excised from the gel and purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega).Enrichment was performed using eight biotinylated oligonucleotides {(AAAC) 6 (AAAG) 6 (AATC) 6 (AATG) 6 (ACCT) 6 (ACAG) 6 (ACTC) 6 (ACTG) 6 } and streptavidincoated magnetic beads (Streptavidin Magnesphere Paramagnetic Particles, Promega).Clones containing inserts were sequenced using the DYEnamic ET Terminator Cycle Sequencing kit (GE Healthcare) and products were resolved on an ABI Prism 377 automated sequencer (Applied Biosystems).The microsatellite-like sequences were analyzed using Tandem Repeats Finder (Benson, 1999) and flanking primers were then designed using the Primer 3 software (Rozen and Skaletsky, 2000).
To analyze if loci were variable up to 30 individuals from two collection sites in the São Francisco River (Brazil) were used.All loci were amplified following the PCR method described by Schuelke (2000), in which three primers are used simultaneously, a fluorescently-marked M13 primer, a forward primer with a fusion of a leading M13 universal sequence, and a reverse primer.PCR reactions were carried out in 10 μL reactions containing 100 ng of DNA, 200 μM dNTPs, PCR buffer (20 mM Tris-HCl, pH 8.4, and 50 mM KCl; LGC Biotecnologia), 4 pmol of each reverse and 6-FAM or NED M13 (-21) primers and 1 pmol of the forward primer, 1.5 mM MgCl 2 and 1 U of Taq DNA Polymerase (LGC Biotecnologia).Conditions of the PCR amplification were as follows: 1 cycle at 95 °C (5 min), 35 cycles at 94 °C (30 s), 30 s at the annealing temperature (Table 1) and 72 °C (30 s), followed by 8 cycles 94 °C (30 s), 53 °C (45 s), 72 °C (45 s), and a final extension at 72 °C for 10 min.
For genotyping, the PCR products were subjected to electrophoresis along with GeneScan ROX 350 (Applied Biosystems) internal size standards on an ABI 377 automated sequencer (Applied Biosystems).Allele sizes were assigned using the Genescan and Genotyper 2.5 software packages (Applied Biosystems).Hardy-Weinberg equilibrium (HWE), observed and expected heterozygosities and linkage disequilibrium tests were performed using the software Genepop 3.3 (Raymond and Rousset, 1995).
Of the 96 sequenced clones, 51 (53.13%) contained microsatellite sequences and primers were designed for 31 loci (Par 55 -Par86).Of these, 15 were successfully amplified.Only loci Par61, Par72, Par73 and Par74 presented a monomorphic pattern, i.e. the most common alleles presented a frequency superior to 95% (Zou et al., 2005), whereas the others showed high levels of polymorphism.The number of alleles per polymorphic locus varied from 5 (Par76) to 21 (Par85), revealing a total of 116 alleles.The values of observed and expected heterozygosities ranged from 0.629 (Par69) to 0.926 (Par85 and Par86) and from 0.643 (Par66) to 0.931 (Par80), respectively.Significant deviations from the Hardy-Weinberg equilibrium were found in the Par71, Par83 and Par86 loci (p < 0.05), which may be explained by the presence of null alleles, mating systems, cryptic population structure and non-random sampling.Summary data for all loci are presented in Table 1.
To test cross-species amplification, three individuals of P. costatus and three individuals of P. lineatus were used.In addition to the loci isolated in the present study, 13 previously described loci for P. argenteus (Barbosa et al., 2006) were also amplified in these species.The markers de-358 Barbosa et al.A description of these novel microsatellite loci in P. argenteus and the data showing their heterologous amplification in P. costatus and P. lineatus will provide useful markers that will allow a better understanding of the Prochilodus population structure, and will aid future P. argenteus genetic mapping studies.

Table 1 -
Characterization of microsatellite loci in Prochilodus argenteus.forP.argenteus amplified and were polymorphic in both species tested.The efficiency of heterologous amplifications was 100% in P. costatus and 95.45% in P. lineatus (Par34 presented a faint band).Allele sizes for P. costatus and P. lineatus corresponded to the size range observed for P. argenteus.In P. costatus the loci Par34 and Par76 were monomorphic, while Par35, Par69, Par71, Par76 and Par82 were monomorphic in P. lineatus.All other remaining loci were polymorphic in their respective species (Table2).
a , total number of different alleles observed; H O , observed heterozygosity; H E , expected heterozygosity; HWE test, p values for Hardy-Weinberg equilibrium test; *p < 0.05.veloped