In present study, we have developed eight polymorphic microsatellite markers that are specific for M. barbata. Genomic DNA was extracted from muscle tissues preserved in 95% ethanol by following standard phenol-chloroform procedure [19]. The enrichment of DNA fragments was carried out using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method [20] with a minor modification [21]. This PIMA (PCR isolation of microsatellite arrays) approach has been proposed [20]. It takes advantage of the fact that the RAPD fragments contain microsatellite repeats more frequently than random genomic clones [22]. Several RAPD primers were selected to amplify the target DNA fragments. The RAPD-PCR amplifications were performed in a thermal cycler (Bio-Rad) with a reaction mixture (50 μL) containing 20–100 ng DNA, 0.2 mM of each dNTP, 2 mM MgCl₂, 0.5 U Taq polymerase (Promega), and 5 pmol of one RAPD primer. The PCR program were as follows: initial denaturing for 3 min at 95 °C for 1 cycle, 40 cycles of 1 min at 94 °C, 45 s at 42 °C, 2 min at 72 °C, followed by 10 min at 72 °C for an additional extension step. Approximately 100 ng of PCR product was ligated into a pGEM-T vector (Promega) according to the manufacturer’s instructions. The ligation mixture was then transformed into Escherichia coli competent cells to form the enriched microsatellite sequence library. Colonies of RAPD fragments were analyzed to verify existence of repetitive sequences. Clones were screened using repeat-specific and vector primers [20]. In positive clones, the repeat-specific and vector primers amplified DNA fragments that contain microsatellites, whereas no amplification was found in negative clones. Plasmid DNA from positives was purified using the High-Speed Plasmid Mini Kit (Geneaid). Both strands of the DNA insert were sequenced. DNA sequencing in both directions was conducted with an Applied Biosystems ABI3730 automated sequencer (Applied Biosystems). Primers for these loci were designed using PRIMER 3 software [23] and synthesized. Primers were designed according to the nucleotide sequences upstream and downstream of the repetitive DNA. A total of 8 primer pairs were designed from 8 sequences as the remaining sequences were too close to the cloning site. Polymorphisms of these microsatellite loci were assessed by 40 M. barbata individuals collected from Fujian Province, China and PCR conditions were optimized for each pair of primers. Reactions were performed in a total volume of 15-μL containing 10 ng of genomic DNA, 0.2 mM dNTP, 2 mM MgCl₂, and 0.12 μM of each primer. The PCR program consisted of 94 °C for 5 min followed by 35 cycles at 94 °C for 30 s, 30 s at primer-specific annealing temperature (Table 1), 72 °C for 45 s and a final extension step at 72 °C for 5 min. Individuals were genotyped on 6% denaturing polyacrylamide gels stained with ethidium bromide straining and sized by comparison to a 10-bp DNA ladder standard (Invitrogen).

Genotype data files were inter-converted for the various analytical software programs using CREATE [24] to minimize errors. The allele number, size range, number of bands per individual, expected (H_E), and observed heterozygosity (H_O) were quantified using the Arlequin version 3.5 [25]. Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were performed using GENEPOP 3.4 software [26]. Results of tests for linkage and Hardy–Weinberg disequilibria were corrected for multiple comparisons by applying sequential Bonferroni corrections [18]. Inbreeding coefficient (F_IS) [27] and the significance of these values were calculated for each population with GenePop Web Version 4.0.10 [26,28].