Sau3ai

Six genome-wide CAT-associated SNPs (rs1107877, rs11185115, rs12202737, rs12626864, rs13729, and rs9825563) were experimentally confirmed in Chinese populations. Genomic DNA was extracted from whole blood using the standard phenol/chloroform method. Among them, rs11185115 within NTNG1 was replaced by a nearby SNP, rs1444041 (r² = 0.954), which was not in the original dataset, owing to a lack of the appropriate SNPstream primers; rs12202737 was represented by rs13729 from the same ULBP3 gene because of their complete LD in the CHB population (r² = 1). In total, four SNPs (rs12626864, rs13729, rs1444041, and rs9825563) were genotyped using the Beckman SNPstream genotyping system [63] (link), while one SNP (rs1107877) was genotyped by the PCR-RFLP method (Sau3AI, Takara, China). For 2 UVR-associated SNPs, rs1586360 was not genotyped owing to its low MAF in the CHB populations (MAF < 0.1). Only rs7531583 was genotyped using the same Beckman SNPstream genotyping system.

E. coli competent cells (DH5α and BL21 (DE3)) were bought from Nanjing Vazyme Biotech Co., Ltd. Plasmids, including pET-28a and pUC118 vector, were obtained from Miaoling Biotechnology Co., Ltd. (Wuhan, China). Reaction enzymes (restriction endonucleases including Sau3AI, NcoI, HindIII, and T4 ligase) and DNA/protein markers were procured from Takara Bio (Kusatsu, Japan). All bacterial strains were grown in a commonly used Luria–Bertani (LB) broth at appropriate temperatures.

Cloning of the Peace gene (Peace genomic sequence, GenBank/EMBL/DDBJ accession AB897866) from genomic DNA was carried out following the directions for the Stratagene phage cloning system (Stratagene, USA). Total DNA extracted from V2 petals was partially digested with Sau3AI (TAKARA, Japan). After a partial fill-in reaction, total DNA fragments were ligated with lambda FIX/XhoI partial fill-in treated DNA (Stratagene, USA), and a packaging reaction was carried out using GigaPack Gold (Stratagene, USA). Recombinant phages were selected by plaque hybridization with Peace cDNA (clone cPpP14) as a probe. Plaque hybridization was carried out using an ECL non-radioactive DNA labelling and detection kit (GE Healthcare, UK).

For RFLP analysis, the COI barcode fragment (Table 1) was digested with HindIII (Takara) to distinguish between D. dentex and other species (Table 3) and with Sau3AI (EnzyQuest) to identify P. pagrus. Additionally, for P. pagrus and related species, the cytb fragment (Table 1) was digested with Sau3AI, and a 1292 bp fragment of the CR (Table 3) amplified with cytbF2 and CRR primers was digested with XbaI (Takara).
The products were run on 1.2% agarose gels stained with Midori Green (Nippon Genetics), and the band sizes were estimated with FastGene^® 100 bp DNA Ladder (Nippon Genetics) and 50 bp DNA Ladder (Jena Bioscience GmbH).