Genemapper 4

A total of 366 polymorphic microsatellite markers (data provided upon request), representing 22 autosomes and X chromosome at approximately 10 cM intervals, were applied for genome-wide linkage screening. Twelve additional microsatellite markers including D14S122, D14S1023, D14S1070, D14S283, D14S972, D14S64, D14S1032, D14S1068, D14S976, D14S978, D14S139 and D14S1057 were further selected for fine mapping on chromosome 14. PCR amplifications (Applied Biosystems) of the microsatellite loci were carried out using fluorescently labeled primers according to a previously described protocol^{7 (link)}. Genotyping data were collected using GeneMapper 4.1 (Applied Biosystems).

The genotypes of BTLA rs16859629, rs1982809, rs2171513 and rs3112270 SNPs were analyzed using a SNPscan Kit (Genesky Biotechnologies Inc., Shanghai, China) as described previously [22–24 (link)]. PCR process was conducted in a 20-μl mixture volume in 96-well plates. ABI 3730xl DNA Analyzer was used to identify the genotype. The data of the sequencing were read by GeneMapper 4.1 (AppliedBiosystems, U.S.A.). One hundred and eleven DNA specimens were randomly chosen for repeat genotyping by another person in a blind fashion, and the obtained variants were concordant.

SNaPshot extension primers for detection of kdr west (L1014F) and kdr east (L1014S) mutations, A296G rdl (Ala-Gly) and A296S rdl (A296S) mutations, and G119S ace-1 (Gly-Ser) mutation were designed (Table 2) to anneal on the sense strand immediately adjacent to the mutation site. Each extension primer was synthesized with a different length of poly (dT) tail to allow separation of SNaPshot products on the basis of size. SNaPshot analysis was performed using an Applied Biosystems SNaPshot Multiplex Kit (Applied Biosystems Co., Ltd., USA). Extension reactions were performed in a volume of 10 μl containing 5 μl of SNaPshot Ready Multiplex Ready Reaction Mix, 1 μl of extension primer mix (Table 2) and 2 μl of ddH₂o. Thermal cycler conditions were: 96°C for 1 min; 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Each 10 μl of extension products were purified with 1 unit of SAP at 37°C for 1 h and 75°C for 15 min. Then, 0.5 μl of the purified extension products were mixed with 0.5 μl of Liz120 size standard, and 9 μl of Hi-Di™ formamide, and were denatured at 95°C for 5 minutes then sequenced using ABI3730XL. Analysis was performed using GeneMapper 4.1 (Applied Biosystems Co., Ltd., USA).

Venous blood samples were obtained as part of the health examination and were anonymized. Samples were collected in EDTA-containing tubes from each participant following a 12-h fast. Genomic DNA was purified from the samples using a whole blood genome extraction kit (Tiangen Biotech, Beijing, China) and stored at −20 °C until use. Tag single nucleotide polymorphisms (SNPs) of 5-HTR2A in the Chinese Han population were identified in the Haplotype Map database (National Center for Biotechnology Information, Bethesda, MD, USA). The SNPs rs6313, rs1923884, and rs2070040 were genotyped with the SNaPshot SNP assay. Data were analyzed using GeneMapper 4.1 (Applied Biosystems, Foster City, CA, USA).

DNA of each fish was isolated with the method described earlier [39 (link)]. A total of 144 polymorphic microsatellite (MS) markers covering the 22 linkage groups (LG) of the tilapia genome near-evenly were selected from a published linkage map [25 (link)]. Detailed information about these markers is provided in S1 & S2 Tables. PCR amplification using fluorescently labeled primers [25 (link)] was carried out for each sample in a 25 μL reaction volume containing 10 ng genomic DNA, 1 x PCR buffer containing 1.5 mM MgCl₂, 0.2 μM dNTPs, 0.2 μM forward and reverse primers, and 0.5 units of Taq DNA polymerase (Finnzymes, Espoo, Finland). The PCRs were performed in PTC-100 thermal cycler (MJ Research, CA, USA) under the following conditions: 2 min denaturation at 94°C; 35 cycles of 30 s at 94°C, 30 s at 55–60°C and 30 s at 72°C and a final extension at 72°C for 10 min. The PCR products were resolved on an ABI3730xl DNA Genetic Analyzer (Applied Biosystems, Foster City, USA). Fragment sizes were analyzed against the internal size standard of GeneScan-500 ROX (Applied Biosystems, Foster City, USA) using GeneMapper 4.1 (Applied Biosystems, Foster City, USA). Our genotyping system is able to differentiate size difference of one base pair among alleles. The genotypes were exported to an Excel table for further data analysis.