Obscure puffer (Takifugu obscurus) is an anadromous fish species, mainly inhabiting South of China. They usually grow in the sea before sexually mature, then return to fresh water to spawn, and they are famous for puffing behavior, powerful toxins in the internal organs, and edible muscle (Akira et al. 2005). Puffer fish are considered to be good model animals and the genome project of T. rubripes was completed in 2002 (Aparicio et al. 2002). The wild resource of T. obscurus has sharply decreased under the effect of overfishing and water pollution. Documents about T. obscurus mostly focused on the physiology (Yan et al. 2004) and mitochondrial DNA (Shao et al. 2006). There are no molecular markers such as microsatellite isolated and application in this species reported to date, in spite of many molecular markers including AFLP, SSR have been developed and used broadly in other fish species (Liu et al. 2003; Chen et al. 2007). Lack of enough polymorphic microsatellite markers has limited the development of genetic diversity, population structure, and molecular marker-assisted breeding in this species.

In the present study, we developed 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of T. obscurus and tested their applicability to additional three fish species including redfin puffer (Takifugu rubripes), tawny puffer (Takifugu flavidus) and yellowfin puffer (Takifugu xanthopterus).

A sample of 30 individuals of T. obscurus were selectively collected in Nanjing city, China. Genomic DNA was extracted from the fin tissue as described by Ma et al. (2007). A dinucleotide-enriched genomic library was constructed using the fast isolation by amplified fragment length polymorphism (AFLP) of sequences containing repeats (FIASCO) protocol (Zane et al. 2002), and the detailed procedure was described by Liao et al. (2007). In brief, the whole genomic DNA was digested with Mse enzyme (New England Biolabs) and ligated to the adapters (Oligo A: 5′ HO-TACTCAGGACTCAT-OH 3′ and Oligo B: 5′ HO-GACGATGAGT CCTGAG-OH 3′). Bio-labelled probes (GT)13 were used to hybridize with the recovered products of pre-amplification (the size ranged from 300 to 900 bp). Subsequently, the hybrids were captured by streptavidin-coated magnetic beads (Promega Corporation), and the final DNA fragments eluted from the magnetic beads were amplified using the corresponding primers. The amplification products were directly cloned into pMD-18T vector as described by Chen et al. (2004) and 60 recombinant clones were randomly selected for sequencing using ABI Prism 3730 automated DNA sequencer (PE Corporation), of which 55 clones containing microsatellite repeats. Finally, PCR primers were designed for 20 loci using the software Primer Premier 5.0, of which 10 loci showing polymorphic. These microsatellite sequences have been deposited in GenBank (EU391391–EU391400).

Polymerase chain reaction (PCR) were performed on a Peltier Thermal Cycler (PTC-200) in 25 μl total volume that included 0.4 μM each primer, 0.2 mM each dNTP, 1× PCR buffer, 1.5 mM MgCl2, 0.75 unit Taq polymerase, and approximately 100 ng template DNA under the following conditions: one cycle of denaturation at 94°C for 4 min; 30 cycles of 30 s at 94°C, 50 s at a primer-specific annealing temperature (Table 1), and 50 s at 72°C. As a final step, products were extended for 7 min at 72°C. The PCR products were separated on 6% denaturing polyacrylamide gel, and visualized by silver-staining. The size of alleles were estimated according to the pBR322/Msp marker (TianGen Biotech CO., LTD.). Genetic diversity indexes were calculated using POPGENE version 1.31 (Yeh et al. 1999) software. Significance values for all multiple tests were corrected by sequential Bonferroni procedure (Rice 1989).

Table 1 Characterization of 10 polymorphic microsatellite loci in obscure puffer (T. obscurus)
Full size table

Ten of the 20 tested microsatellite loci were polymorphic in 30 individuals of T. obscurus (Table 1). The number of alleles per locus ranged from 4 to 10, and observed and expected heterozygosity ranged from 0.57 to 0.86 and from 0.68 to 0.90, respectively. Three loci (Tob11, Tob61 and Tob107) showed significant departures from Hardy–Weinberg equilibrium after sequential Bonferroni correction (P < 0.005). Such deviations might be caused by the limited sample size used in our test or the presence of null alleles confirmed by MICRO-CHECKER version 2.2.3 software (Van Oosterhout et al. 2004), but no evidences for stuttering and allelic dropout were found in all loci. No significant genotypic linkage disequilibrium (LD) was found between all pairs of the 10 loci after Bonferroni correction (P > 0.005).

The 10 polymorphic microsatellite loci were also tested for cross-amplification in additional three fish species including redfin puffer (T. rubripes), tawny puffer (T. flavidus) and yellowfin puffer (T. xanthopterus) (Table 2). Eight or seven individuals of each species were tested and PCR was carried out under the conditions as described previously. The result showed high degree of polymorphism of these loci in the three fish species. All 10 loci were polymorphic in each fish species, and the number of alleles per locus ranged from two to six in the three fish species.

Table 2 Cross-species amplification of 10 microsatellite loci in additional three fish species including redfin puffer (T. rubripes), tawny puffer (T. flavidus) and yellowfin puffer (T. xanthopteru s)
Full size table

To the best of our knowledge, it is the first time to report polymorphic microsatellite markers in T. obscurus that will allow studies of the population structure and genetic diversity of T. obscurus in the future.