Microsatellites from B.sinensis were isolated using a modified enrichment technique described by Ding [3]. Genomic DNA was extracted from the muscle tissue using a standard traditional phenol-chloroform procedure [4] and digested with Sau3AI at 37 °C overnight. Fragments from 500–2000 bp were excised from agarose gels using a QIAqucik Gel Extraction Kit (QIAGEN) and ligated to two oligo adapters (Oligo A 5′-GATCGTCGACGGTACCGAATTCT-3′ and Oligo B 5′-GTCAAGAATTCGGTACCGTCGAC-3′) to facilitate amplification by PCR. The amplified genomic fragments were subsequently hybridized with the 5′-biotinylated oligo probes ATA(CA)22C and fragments containing potential repeat motifs were captured with steptavidin-coated magnetic beads (Dynabeads^® M-280, Invitrogen). To increase the amount of potential repeat motifs, a “recovery” PCR was performed using oligo B as the PCR primer. The PCR products were then purified and ligated into PMD19-T vector (TAKARA) and transformed into DH5α competent cells. Cells were then plated onto LB agar, X-gal and ampicillin and incubated overnight at 37 °C.

The positive clones were identified by PCR with vector-specific primers. After being identified, 196 positive clones were randomly selected for sequencing using ABI3730XL sequencer (Applied Biosystems). Chromatograms were assembled and edited using SEQUENCHER 4.9 (Gene Codes Corporation), and 70 primers were designed for each unique amplicon containing a target microsatellite repeat. Initially, eight samples from different localities were used to test amplification of loci and evaluate polymorphic content. The PCR amplification was performed in 15 μL volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂ 0.2 mM each dNTPs, 0.4 μM each primer, 1 U Taq polymerase (TAKARA) and 30 ng genomic DNA. After denaturation for 5 min at 94 °C, followed by amplication for 30 cycles (94 °C for 30 s, annealing temperature for each pair of primers (Table 1) for 30 s, 72 °C for 30 s) and a final step at 72 °C for 5 min. PCR products were mixed with the GS500LIZ size standard (Applied Biosystems) and formamide and run on an ABI3130xl DNA Analyzer. Fragment analysis and genotyping were performed using Genemapper version 4.0 (Applied Biosystems). Out of the 70 primer pairs tested, 20 pairs were successfully amplified by PCR and further characterized using additional samples at Chiku Lagoon (23°55′05″ N-120°02′57″ E) from Taiwan (n = 30).

The number of alleles, observed and expected heterozygosities, P value of Hardy-Weinberg and linkage disequilibria were estimated by using POPGENE [5].