All the samples were bred in a laboratory for at least one week. Then, the samples negative to S. japonicum were selected. After removal of the gut and digestive glands from the soft parts of the snail, genomic DNA was extracted from the muscle tissues of the snail followed by the standard DNA extraction procedure using mollusk DNA Kit (Omega, Norcross, GA, USA) [17]. Then, we followed the protocol of Hammond for construction of a microsatellite enriched genomic library, with some minor modification [18]. Briefly, total genomic DNA was digested with restriction enzyme Sau3AI (Fermentas, Burlington, Ontario, Canada) and then ligated to Sau3AI AFLP adaptor followed by amplification with adaptor-specific primers (SauLA: 5′-GCG CTA CCC GGG AAG CTT GG-3′, and SauLB: 5′-ATC CCA AGC TTC CCG GGT ACC GC-3′).

Microsatellite enrichment involved three rounds of PCR amplification. The first enrichment PCR used SauLA sequence as primer and ligated DNA as template. The amplified DNA fragments were then denatured and hybridized with biotinylated oligonucleotides [(AAT)17, (GA)25, (CCT)17, (AC)25, (CAG)17, (CAC)5, (TC)10, (TG)18]. The target moleculars bond with complementary microsatellites in the genomic library. The genomic fragments with microsatellite were captured with Vectrex Avidin D through hybridization. Genomic DNA that contained microsatellite repeats were stripped from Vectrex Avidin D and concentrated by ultrafiltration using Amicon Ultra-4 (Millipore). The second enrichment was identical to the first enrichment procedure to further amplify genomic fragments with microsatellites. The third enrichment procedure contained PCR amplification only. The PCR used the SauLA sequence as primer and concentrated DNA from the second enrichment as template. The amplified fragments were cloned into a plasmid using TOPO TA Cloning Kit (Invitrogen), and a microsatellite-enriched genomic library was thus constructed.

Thirty sequences were chosen and used to amplification. Primers were designed flanking each suitable microsatellite sequence, using the primer 3.0 computer program [19]. A total of 21 primer pairs produced successful and consistent amplification, and those primers were further examined for polymorphism with O. hupensis field snails. One of the two primers that were used to amplify each locus was labeled with a fluorescent dye such as HEX, NED and FAM. Polymorphisms of microsatellite loci were evaluated in 80 wild individuals O. hupensis from Fujian, Sichuan, Guangxi and Yunnan province. Microsatellites were amplified under the following conditions. The reaction mixtures (25 μL) total containing 1× Taq buffer, 0.15 mM dNTPs, 0.5 μM forward and reverse primers, 1.5 mM MgCl₂, 1.0 U Taq polymerase (Tiangen, Beijing, China) and about 20 ng gemonic DNA. PCR amplification was carried out on a Thermal Cycler (PTC-100, BIO-RAD, USA). Conditions included the following steps: an initial denaturation at 95 °C for 5 min, 35 cycles of 95 °C for 1 min, annealing at a set temperature depending on each locus (Table 1) for 45 s and 72 °C for 1.5 min, and final extension at 72 °C for 5 min. PCR products were determined using the Genetic Analyser 3730 (Applied Biosystems, Carlsbad, CA, USA), and analyzed by genescan 3.7 and genotyper 3.7 (Applied Biosystems).

Standard genetic diversity parameters of polymorphic loci, e.g., the number of alleles (N_A), and expected (H_E) and observed (H_O) heterozygosity, and Hardy-Weinberg equilibrium (HWE) and linkage dis-equilibrium were tested using GENEPOP 4.0 [20]. Null allele frequencies were calculated using Micro-Checker 2.2.3 [21].