Abi 3730 platform

The DNA samples were amplified for identification as described in the previous study (Lin et al., 2020 (link)). The universal cytochrome oxidase subunit (cox) I primer set for rotifer identification was used: LCO1490 5′-GGTCAACAAATCATAAAGATATTGG-3′ and HCO2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′. The PCR amplification system for the target gene comprises 1 μl of cDNA, 12.5 μl of a mixture, 1 μl of forward primer, 1 μl of reverse primer, and 9.5 μl of double deionized water. The PCR cycling conditions were carried out: initial denaturation step at 94°C for 5 min followed by 30 cycles of 94°C for 45 s, 48°C for 45 s, and 72°C for 45 s with a final extension step at 72°C for 10 min. In addition, The universal coxI primer set was also used for the Biomphalaria species identification. The PCR conditions for the marker amplification were performed: denaturation at 94°C for 5 min, 30 cycles of 94°C for 50 s, 55°C for 50 s, 72°C for 50 s, and final extension at 72°C for 10 min. The PCR products were detected on 3% agarose gel electrophoresis and purified according to the protocol of the Qiagen gel extraction kit (Qiagen, Germany). The purified PCR products were sequenced on an ABI-3730 platform (Applied Biosystems) by the Majorbio company (Guangzhou, China).

To validate the putative SNPs identified in transcripts, the same cDNA samples as for the transcriptome profiling (pool of eighteen wild Portunus trituberculatus) were used. Twenty transcripts containing 56 potential SNPs and sufficient flanking regions were randomly selected for primer design. PCR products were sequenced directly in both directions with forward and reverse primers using Sanger technology on the ABI3730 platform (Applied Biosystems). Sequencing chromatograms were visually analyzed with Chromas2.32 (Technelysium Pty. Ltd.), and SNPs were identified as overlapping nucleotide peaks.